CN102203605B - Devices and methods for processing biological samples - Google Patents

Abstract

The present invention provides systems, devices, apparatus and methods for automated bioprocessing. Examples of protocols and biological treatment procedures suitable for the present invention include, but are not limited to: immunoprecipitation, chromatin immunoprecipitation, recombinant protein isolation, nucleic acid separation and isolation, protein labeling, separation and isolation, cell separation and isolation, food safety analysis, and bead-based automated separation. In some embodiments, the invention provides automated systems, automated devices, automated cassettes, and automated methods for Western blot processing. Other embodiments include automated systems, automated devices, automated cassettes and automated methods for isolating, preparing and purifying nucleic acids, such as DNA or RNA or fragments thereof, including plasmid DNA, genomic DNA, bacterial DNA, viral DNA and any other DNA, and automated systems, automated devices, automated cassettes and automated methods for processing, isolating and purifying proteins, peptides, and the like.

Description

Devices and methods for processing biological samples

field of invention

The present invention relates to devices and methods for processing biomolecules, and more particularly to automated methods and devices for processing biomolecules.

Background of the invention

Certain laboratory procedures are still largely performed by inefficient manual methods that require the individual attention of the scientists or laboratory technicians who are performing the procedures. Many of these procedures would benefit from automation. For example, nucleic acid purification, such as plasmid preparation, is a time-consuming and inefficient task that is not currently automated. Incremental improvements, such as the introduction of precipitation filters, have reduced the hands-on time required, yet even the most advanced nucleic acid purification kits still require hours and individual attention. Likewise, Western blot analysis (Western blot analysis) processing is a labor-intensive process that tethers a scientist or lab technician to the bench during the process. Furthermore, such processes suffer from human error and lack repeatability in a manually intensive procedure. To some extent, what is needed and provided herein is a compact, inexpensive, user-friendly and flexible instrument for reactions on solid supports, preproteomics sample preparation, nucleic acid applications and cell separation applications with enhanced ease of use, reduced labor time, reduced errors, and enhanced reproducibility.

Summary of the invention

In some embodiments, the present invention provides systems, devices, devices and methods for automated bioprocessing. Examples of protocols and bioprocessing procedures suitable for the present invention include, but are not limited to: immunoprecipitation, chromatin immunoprecipitation, recombinant protein isolation, nucleic acid isolation and isolation, protein labeling, isolation and isolation, cell isolation and isolation, and bead-based automated separate. In specific embodiments, the present invention provides automated systems, automated devices, automated cartridges and automated methods for Western blot processing. Other embodiments include automated systems, automated devices, automated cassettes and automated methods for isolating, preparing and purifying nucleic acids and automated systems, automated devices, automated cassettes and automated methods for processing, isolating and purifying proteins, peptides, etc., Such nucleic acids such as DNA or RNA or fragments thereof include plasmid DNA, genomic DNA, bacterial DNA, viral DNA and any other DNA or fragments thereof.

In some embodiments, an automated bioprocessing system includes a bioprocessing device, at least one bioprocessing cartridge, a fluid supply, and a device for controlling at least one parameter associated with bioprocessing using the bioprocessing cartridge computer control system. In some embodiments, an automated bioprocessing system can include a plurality of bioprocessing cassettes.

In some embodiments, the automated bioprocessing apparatus comprises one or more cartridge slots, each configured to accommodate a bioprocessing cartridge; a removable fluid container tray comprising a plurality of configurations a fluid container holder capable of holding a fluid container for use during bioprocessing; and a computer control system configured to control at least one parameter associated with bioprocessing in the one or more bioprocessing cartridges.

In some embodiments, an automated bioprocessing device includes a fluid manifold configured to fluidly connect one or more fluid containers in a fluid container holder to one or more process fluid connectors. fluid connector). In some embodiments, the automated bioprocessing apparatus can include a fluid manifold configured to connect one or more control fluid connectors on the bioprocessing cartridge to one or more control fluid supplies. (control fluid supply) connection.

In some embodiments, the automated bioprocessing cartridge comprises at least one bioprocessing chamber configured to house a solid support, the cartridge further comprising a plurality of mesoscale and/or microscale fluid flow A fluid flow channel is in fluid communication with the biological processing chamber via at least one pump.

In some embodiments, the automated method of bioprocessing comprises providing a bioprocessing cartridge containing at least one bioprocessing chamber and a plurality of mesoscale and/or microscale fluid flow channels in fluid communication with the bioprocessing chamber, and At least one process fluid is pumped through at least one of the plurality of mesoscale or microscale fluid flow channels and into a bioprocessing chamber containing a solid support.

In some embodiments, provided herein are bioprocessing cartridges containing at least one bioprocessing chamber, a plurality of mesoscale and/or microscale process fluid channels in fluid communication with the bioprocessing chamber, and a plurality of built-in A pump, the bioprocessing chamber configured to house the blotting membrane, the pump configured to pump fluid through the process fluid channel. Process fluid channel diameters can vary from about 10 μm to about 10 mm, about 100 μm to about 5 mm, about 250 μm to about 2.5 mm, about 500 μm to about 2 mm, or about 1 mm in diameter. The box may be a plastic box. The cassette may also contain an inflexible tube. In some embodiments, the cartridge includes at least one access valve located in at least one defined flow path of the plurality of process fluid channels. Each of the at least one inlet valve may be located in the flow path between at least one process fluid connector and at least one of the plurality of process fluid channels, each process fluid connector configured to fluidly connect the cartridge to a or multiple fluid containers. In some embodiments, the cassette may include more than one inlet valve, wherein each inlet valve may be placed in the flow path between a separate process fluid connector and one of the plurality of process fluid channels, each independently The process fluid connector is configured to fluidly connect the cartridge with one or more fluid containers. The process fluid connector can be a plurality of connectors, for example, 2-20 suction tubes (aspiration tube) and/or extraction tube (expiration tube), 2-10 suction tubes and/or extraction tubes or 4-8 suction and/or extraction tubes. In some embodiments, the cassette can include an inlet valve in each flow path between each process fluid connector and each of the plurality of process fluid channels. In some embodiments, the cassette can include an inlet valve in each flow path between each process fluid connector and each of the plurality of process fluid channels. In some embodiments, the cartridge can include a blotted membrane located in a bioprocessing chamber. The blotting membrane may be a Western blotting membrane. The shape of the box can vary. In some embodiments of the cassette, the depth may vary from about 2 mm to about 5 cm, about 4 mm to about 4 cm, about 5 mm to about 2 cm, about 8 mm to about 1.5 cm, about 9 mm to about 1.2 cm, about 1 cm or 1cm. The box height may vary from about 10 cm to about 25 cm, about 12 cm to about 20 cm, about 14 cm to about 18 cm, about 15 cm to about 17 cm, about 16 cm or 16 cm. The maximum variation range of the box width is about 10 cm to about 25 cm, about 12 cm to about 22 cm, about 16 cm to about 20 cm, about 17 cm to about 19 cm, about 18 cm, 18 cm or about 18.2 cm or 18.2 cm. In some embodiments, the cassette may be rectangular or square. The process fluid connectors may vary in diameter from about 10 μm to about 10 mm, from about 100 μm to about 5 mm, from about 250 μm to about 2.5 mm, from about 500 μm to about 2.0 mm, or about 1 mm in diameter. In some embodiments, the cassette may be packaged. In some embodiments, the diameter of at least one process fluid connector tapers to a smaller inner diameter at its distal end. In some embodiments, at least one process fluid connector closest to (closest to) the process fluid channel has an inner diameter of about 0.5 mm to about 15 mm, about 1 mm to about 10 mm, about 2 mm to about 6 mm, about 4 mm, or 4 mm, and The inner diameter of the at least one process fluid connector end farthest (distal) from the process fluid channel is from about 10 μm to about 10 mm, from about 100 μm to about 5 mm, from about 250 μm to about 2.5 mm, from about 500 μm to about 2.0 mm, or dia. About 1mm. The cartridge may comprise any combination of elements provided in this specification.

Provided herein in some embodiments is an automated blot processing apparatus comprising: one or more cartridge wells, each well configured to receive a bioprocessing cartridge; and an automated control system configured to control at least one and Follow the steps associated with processing the blotted membrane in the bioprocessing cartridge according to the blotting protocol. In some embodiments, the device may include about 1 to about 8, about 2 to about 6, about 3 to about 5, or 4 cartridge slots. In some embodiments, the device can also include a bioprocessing cartridge. The bioprocessing cartridge may comprise a blot, wherein the blot is a western blot, and the blot protocol is a western blot processing protocol. In some embodiments, the blot processing device can also include a blot membrane in a bioprocessing cartridge. The automated control system may be configured to automatically control a maximum of steps, all steps or all but one, two, three or four steps of a blot processing protocol, such as a western blot processing protocol. In some embodiments, an automated blot processing apparatus may include an automated control system configured to eliminate the need for all steps of a blot processing protocol, such as a western blot processing protocol, eliminating the need for all but one, two, or three steps . The automated blot processing device may be any device of any device embodiment as provided herein. The automated blot processing device can use any of the bioprocessing cartridges described in this specification.

In some embodiments, the automated bioprocessing method comprises: a) inserting at least one bioprocessing cartridge into an automated bioprocessing device, the bioprocessing cartridge comprising: i) at least one bioprocessing chamber containing a solid support therein; and ii ) a plurality of mesoscale and/or microscale channels in fluid connection with the bioprocessing chamber; and b) initiating a bioprocessing protocol on the bioprocessing device, the protocol comprising one or more of the following steps: i) controlling pumps and valves on said bioprocessing cartridges to provide reagents and/or samples from one or more containers to at least one bioprocessing chamber of each of at least one bioprocessing cartridge, ii) controlling said biological pumps and valves on the processing cartridge so that the reagents and/or samples are recirculated through at least one bioprocessing chamber of each of the at least one bioprocessing cartridge; and/or iii) controlling the pumps and valves to remove reagents and/or samples from at least one bioprocessing chamber of each of the at least one bioprocessing cartridge.

In some embodiments, the method comprises a method of applying one or more fluids to a solid support comprising: a) inserting at least one bioprocessing cartridge into an automated bioprocessing device, the bioprocessing cartridge comprising: i) at least a bioprocessing chamber containing a solid support therein; and ii) a plurality of mesoscale and/or microscale channels in fluid communication with at least one bioprocessing chamber; b) implementing a pumping sequence on said cartridge, wherein said The pumping sequence includes one or more fluid addition cycles in which fluid is pumped from one or more containers through at least one of a plurality of mesoscale and/or microscale channels and into at least one cavity.

Provided herein is an automated method of processing a blot comprising a) providing a bioprocessing cartridge of any embodiment of the bioprocessing cartridge described herein, wherein the bioprocessing cartridge contains a blot membrane; and pumping at least one process fluid through the at least one of the plurality of process fluid channels and pumped into the bioprocessing chamber. In some embodiments, the method can be performed by means of any bioprocessing plant as described herein. The method may comprise all steps, all but one step, all but two steps, or all but three steps of the blotting protocol carried out by the device on the blotting membrane.

In some embodiments, automated bioprocessing systems, automated bioprocessing devices, automated bioprocessing cartridges, and automated bioprocessing methods include western blot processing systems, western blot processing devices, western blot processing cartridges, and western blot processing methods.

In some embodiments, automated bioprocessing systems, automated bioprocessing devices, automated bioprocessing cassettes, and automated bioprocessing methods include nucleic acid isolation, purification, and/or collection systems, nucleic acid isolation, purification, and/or collection devices, nucleic acid isolation, purification and/or collection kits and nucleic acid isolation, purification and/or collection methods.

Also provided herein are kits comprising any of the bioprocessing cassettes described herein. The kit may also include one or more tubes, containers, or any other suitable fluid reservoir.

Provided herein is a bioprocessing cartridge comprising at least one bioprocessing chamber configured to accommodate a solid support and a plurality of mesoscale and/or microscale process fluid channels that communicate with the bioprocessing fluid via at least one pump. The processing chamber is in fluid communication, wherein the at least one pump is included in or on the bioprocessing cartridge. In some embodiments, the cassette can include at least one inlet valve positioned in a flow path defined by at least one of the plurality of process fluid channels. Each of the at least one inlet valve may be located in the flow path between a process fluid connector and at least one of the plurality of process fluid channels, each process fluid connector configured to fluidly connect the cartridge to one or more fluid containers. In some embodiments, the cassette may include more than one inlet valve, wherein each inlet valve is placed in the flow path between a separate process fluid connector and each of a plurality of process fluid channels, each A separate process fluid connector is configured to fluidly connect the cartridge to one or more fluid containers. In some embodiments, the process fluid connector can be configured to connect to the fluid container through a manifold. In some embodiments, the process fluid connector is configured to connect to the fluid container through a suction tube and/or a withdrawal tube. In some embodiments, the process fluid connectors may be suction and/or extraction tubes. In some embodiments, the cassette can include an inlet valve in each flow path between each process fluid connector and each of the plurality of process fluid channels. The solid support can be selected from the group consisting of blotting membranes, filter cassettes, filter membranes, filter papers, solid phase extraction cassettes, solid phase extraction discs, multiple beads including magnetic beads, and combinations thereof. In some embodiments, the bioprocessing cartridge includes at least one, at least two, or at least three process valves in the flow path between the pump and the chamber. In some embodiments, at least one of the process valves or the inlet valves includes a regulator that closes the valve and/or opens the valve. In some embodiments, at least one of the process valves and/or the inlet valves may comprise a check valve. In some embodiments, the bioprocessing cartridge may comprise two or more or three or more bioprocessing chambers. In some embodiments, the cartridge may also include a plurality of control fluid channels. In some embodiments, the cartridge may further include a control fluid connector connected to each of the plurality of control fluid channels, each control fluid connector configured to fluidly connect the cartridge to one or more automated control systems . In some embodiments, an automated control system controls opening and closing of process valves and inlet valves, and controls at least one pump. In some embodiments, the cartridge can include a dye chamber in fluid communication with at least one process fluid channel, wherein the material in the dye chamber changes color when in contact with a fluid. In some embodiments, the cartridge may comprise a multi-use cartridge. In some embodiments, the treatment chamber can be configured to provide access to the treatment chamber for a user. In some embodiments, the cartridge is a single-use cartridge.

Also provided herein is an automated bioprocessing apparatus comprising: one or more cartridge slots, each slot configured to receive a bioprocessing cartridge; a removable fluid container tray comprising at least one fluid container holder that holds a container configured to hold a container for bioprocessing; and an automated control system configured to control at least one parameter associated with bioprocessing in the one or more bioprocessing cartridges. The device may comprise 2-8 cassette slots or 2-4 cassette slots. In some embodiments, the cassette well may further comprise: a fluid manifold configured to fluidly connect one or more fluid containers in the fluid container holder with one or more process fluid connectors; and/or A control fluid manifold configured to connect the one or more control fluid connectors with one or more automated control systems. The manifold may be configured to connect one or more control fluid connectors with one or more automated control systems. In some embodiments, the one or more automated control systems include a vacuum supply and an air pressure supply. The vacuum supply and/or the air pressure supply may be included in the device. Alternatively, the vacuum supply and/or the air pressure supply may be external to the device. In some embodiments, the vacuum supply can include a vacuum pump. In some embodiments, the air pressure supply may include a compressor. In some embodiments, the cartridge slot may include a plurality of openings for receiving the suction and/or withdrawal tubes on the cartridge and directing the suction and/or withdrawal tubes into the fluid container within the fluid container holder or guide feature. In some embodiments of the device, the device may include at least one removable fluid container tray comprising a set of fluids configured to provide reagents and/or samples to each bioprocessing cartridge in each cartridge slot. Container holder or storage pool. The set of fluid container holders also includes a container configured to contain fluid from or provide fluid to each of the plurality of bioprocessing cartridges. In some embodiments, the bioprocessing device may include a GUI. In some embodiments, the automated control system can provide control of the pumps and valves on the bioprocessing cartridges in each cartridge tank individually. In some embodiments, the automated control system may provide a user with input of one or more control parameters, user selection of pre-programmed protocols, and/or user creation and storage of protocols.

Also provided herein is a method of using an automated bioprocessing method comprising: providing a bioprocessing cartridge comprising at least one bioprocessing chamber containing a solid support therein and a plurality of mesoscales in fluid communication with the bioprocessing chamber and/or microscale process fluid channels, and pumping at least one process fluid through at least one of the plurality of process fluid channels and into the bioprocessing chamber. In some embodiments, the pumping can include pumping one or more reagents and/or sample into the processing chamber and into contact with the solid support. Additionally, said pumping may comprise circulating at least one of said reagents from a channel proximate an upper portion of said chamber through a pump on said cartridge and into a channel proximate a bottom portion of said chamber. In some embodiments of the method, the pumping comprises pumping at least one process fluid through or across the surface of a filter or membrane into the at least one bioprocessing chamber. In some embodiments, a filter or membrane may comprise a blotted membrane. In some embodiments, the blot can be held by a blot holder in the bioprocessing chamber. In some embodiments, the filter or membrane may comprise a western blot membrane. In some embodiments, the filter or membrane may comprise a cell separation membrane. In some embodiments, the filter or membrane can include a lysate filter. In some embodiments, at least one process fluid can include at least one blocking buffer. The at least one process fluid may comprise at least one antibody and/or at least one washing liquid. In some embodiments of the method, the pumping may comprise: a) adding a blocking buffer to the bioprocessing chamber, b) recirculating the blocking buffer across the blotting membrane to form a blocked blotting membrane, c ) adding at least one antibody solution to the bioprocessing chamber, and d) recirculating at least one antibody solution across said blocked blotting membrane. Recirculating at least one antibody solution through the blocked blotting membrane may comprise: a) recirculating the primary antibody solution through the blocked blotting membrane, b) washing the blotting membrane, c) adding the secondary antibody solution to a bioprocessing chamber; and d) recirculating the secondary antibody solution through the washed blotting membrane. In some embodiments of the method, the pumping may comprise pumping at least one process fluid through at least one solid phase extraction membrane, at least one solid phase extraction cartridge, or at least one solid phase extraction disc into at least one A bioprocessing chamber. In some embodiments of the method, the solid phase extraction membrane, solid phase extraction cartridge, or solid phase extraction disc can include a silicon reversibly binding ligand. In some embodiments, the pumping can include pumping the cell culture medium through a cell separation filter to separate cells from the cell culture medium, wherein the cells are captured on the filter. In some embodiments, the pumping can also include: a) resuspending the captured cells in a resuspension buffer, b) lysing the cells in the lysate to form a lysate, c) neutralizing the lysate; and d) clarifying the lysate. The cells can be resuspended and pumped out of the bioprocessing chamber and into a vessel containing lysate. In some embodiments, clarifying the lysate can include pumping the lysate through a filter or membrane to remove unwanted cellular molecules. In some embodiments of the method, the pumping may further comprise: a) extracting at least one biomolecule in a bioprocessing chamber containing the bound membrane, b) washing the bound membrane; and c) washing the bound membrane off biomolecules. In some embodiments of the method, the pumping can also include precipitating biomolecules in a bioprocessing chamber containing a sedimentation filter. In some embodiments of the method, the method can further comprise pre-treating the sample prior to pumping. In some embodiments, pretreatment can include adding a saline solution to the sample. In some embodiments, the saline solution can be NaCl.

Also provided herein is an automated bioprocessing method comprising: a) inserting at least one bioprocessing cartridge into a bioprocessing device, the bioprocessing cartridge comprising: i) at least one bioprocessing chamber containing a solid support therein; and ii) a plurality of mesoscale and/or microscale channels in fluid communication with said bioprocessing chamber, b) initiating a bioprocessing protocol on said bioprocessing device, said protocol comprising one or more of the following steps i) controlling pumps and valves on said bioprocessing cartridge to provide reagents and/or samples from one or more containers to at least one bioprocessing chamber of each of at least one bioprocessing cartridge, ii) controlling all pumps and valves on said bioprocessing cartridges, thereby recirculating said reagents and/or samples through at least one bioprocessing chamber of each of at least one bioprocessing cartridge; and/or iii) controlling said bioprocessing pumps and valves on the cartridge to remove reagents and/or samples from at least one bioprocessing chamber of each of the at least one bioprocessing cartridge.

Provided herein is a method of applying one or more fluids to a solid support, the method comprising: a) inserting at least one bioprocessing cartridge into a bioprocessing device, the bioprocessing cartridge comprising: i) at least one solid support therein a bioprocessing chamber of the support; and ii) a plurality of mesoscale and/or microscale channels in fluid communication with the bioprocessing chamber; b) implementing a pumping sequence on the cartridge, wherein the pumping sequence Including entering one or more fluid addition cycles, wherein fluid is pumped from one or more containers through a fluid flow channel and into the cavity; wherein the fluid added in any fluid addition cycle is the same as in any other fluid addition cycle The fluids added in the cycle are the same or different. In some embodiments, the pumping sequence can also include entering a purge cycle after each fluid addition cycle, which includes pumping the fluid in the chamber into a designated waste container. In some embodiments of the method, the pumping sequence further includes entering a circulating cycle after any fluid addition cycle, wherein the circulating cycle includes opening a circulator in a fluid flow channel connected to the bottom of the chamber. valve and pump fluid from the bottom of the chamber through one or more fluid flow channels and into the top of the chamber. In some embodiments of the method, the method can further comprise initiating and terminating the pumping sequence with a programmable controller. In some embodiments, the method can also include independently opening and closing a valve in each process fluid connector to selectively control the amount of fluid flowing into or out of the fluid flow channel. In some embodiments, the method may further include inserting a plurality of cartridges into the cartridge slot, and performing a pumping sequence on each cartridge, wherein the pumping sequence performed on each cartridge is the same as that performed on any other cartridge. The pumping sequence is the same or different. The pumping sequence performed for each cassette can be performed simultaneously. In some embodiments, the method can also include initiating and terminating a pumping sequence for each cartridge with a programmable controller. In some embodiments, the controller may be a central processing unit of a computer. In some embodiments, the method can also include storing one or more selection programs in the controller. In some embodiments, the method may also include initiating a specified pumping sequence by selecting a program stored in the controller. In some embodiments, the method can also include displaying the one or more selection programs on the GUI.

Also provided herein is an automated bioprocessing system comprising: a) a bioprocessing apparatus comprising i) one or more cartridge wells, each well configured to receive a bioprocessing cartridge, and ii) an automated control system configured to controlling at least one parameter associated with bioprocessing in the one or more bioprocessing cartridges, and b) one or more bioprocessing cartridges comprising i) at least one bioprocessing chamber configured to accommodate a solid support, ii) A plurality of mesoscale and/or microscale process fluid channels in fluid communication with the bioprocessing chamber via at least one pump included in or on the bioprocessing cartridge.

As used herein, cards, cartridges, bioprocessing cards and bioprocessing cartridges are considered to be interchangeable. Also provided herein is the cassette, device or method of any of the preceding embodiments herein, wherein the blot comprises a protein or nucleic acid.

Brief description of the drawings

The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description and accompanying drawings which illustrate exemplary embodiments which utilize the principles of the invention:

Figures 1A-1D depict alternative bioprocessing device embodiments;

Figures 2A-2C depict rear views of alternative bioprocessing devices;

Figures 3A and 3B show an embodiment of a graphical user interface (GUI);

Figure 4 shows an exploded view of an embodiment of a biological processing unit;

Figure 5 shows an embodiment of the bioprocessing device with the housing removed;

Figure 6 shows an exploded view of an embodiment of a removable fluid container tray;

Figure 7 shows an exploded view of an embodiment of a fluid container holder;

8A-8F show various views and embodiments of reagent trays and reagent reservoirs;

Figures 9A and 9B show perspective views of a cartridge holder embodiment;

Figures 10A and 10B show side views of a cartridge holder embodiment;

Figures 11A and 11B show cross-sectional views of an embodiment of a cartridge holder;

Figure 12 shows an exploded view of an embodiment of a bioprocessing cartridge;

Figures 13A and 13B show front and rear views, respectively, of an embodiment of a bioprocessing cartridge;

Figure 14 shows a perspective view of an embodiment of a bioprocessing cartridge showing the front and back;

Figure 15 shows a detailed view of an embodiment of the connection between the process fluid connector and the suction/extraction tube.

Figure 16A is an exploded view of an embodiment of a bioprocessing cartridge; Figures 16B-16D show close-up views of a portion of the bioprocessing cartridge shown in Figure 16A;

Figures 17A and 17B show front and rear views of an embodiment of a bioprocessing cartridge;

Figures 18A and 18B show front and rear views of an embodiment of a bioprocessing cartridge;

Figures 19A and 19B show internal and external views, respectively, of an embodiment of a bioprocessing cartridge;

Figures 20A and 20B show alternative bioprocessing cartridge embodiments;

Figures 21A and 21B show detailed views of a blotted membrane holder that can be inserted into an embodiment of a bioprocessing cartridge;

Figures 22A and 22B show alternative embodiments of blot holders;

Figure 23 shows a high-level flow diagram of a basic user interface for operating an embodiment of a bioprocessing device;

Figures 24A and 24B show the results of western blots performed as described in Examples 1 and 2;

Figures 25A and 25B show the results of western blots performed as described in Examples 3 and 4;

Figures 26A-26E show the results of western blots performed as described in Examples 5, 6, 7, 8 and 9;

Figures 27A-27D show the results of western blots performed as described in Examples 10A and 10B and Examples 11A and 11B;

Figures 28A and 28B show the results of western blots performed as described in Examples 12A and 12B;

Figures 29A and 29B show the results of western blots performed as described in Examples 13A and 13B;

Figures 30A and 30B show the results of western blots performed as described in Examples 14A and 14B;

Figures 31A-31D show the results of western blots performed as described in Examples 16A and 16B and Examples 15A and 15B;

Figures 32A-32D show the results of western blots performed as described in Examples 18A and 18B and Examples 17A and 17B;

Figures 33A and 33B show the results of western blots performed as described in Examples 19A and 19B;

Figures 34A and 34B show the results of western blots performed as described in Examples 20A and 20B;

Figures 35A and 35B show the results of western blots performed as described in Examples 21A and 21B;

Figures 36A and 36B show the results of western blots performed as described in Examples 22A and 22B;

Figure 37 shows the results of nucleic acid purification performed as described in Example 23;

Figure 38 shows the results of nucleic acid purification performed as described in Example 24;

Figure 39 shows the results of nucleic acid purification performed as described in Example 25;

Figure 40 shows the results of nucleic acid purification performed as described in Example 26;

Figures 41A-41F show the results of western blots performed as described in Example 27;

Figures 42A-42C show the results of western blots performed as described in Example 27;

Figures 43A-43F show the results of a western blot performed as described in Example 27;

Figures 44A and 44B show the results of nucleic acid purification performed as described in Example 30;

Figures 45A and 45B show the results of nucleic acid purification performed as described in Example 31; and

FIG. 46 shows the results of nucleic acid purification performed as described in Example 32.

Detailed description of the invention

Automated bioprocessing systems, automated bioprocessing devices, automated bioprocessing cassettes, and automated bioprocessing methods disclosed herein include automated bioprocessing systems, automated bioprocessing devices, automated bioprocessing systems for implementing one or more protocols for processing biomolecules Bioprocessing cassettes and automated bioprocessing methods, e.g. performing a bioprocessing procedure selected from the group consisting of immunoprecipitation, chromatin immunoprecipitation, recombinant protein isolation, nucleic acid isolation and isolation, protein labeling, isolation and isolation, cell isolation and isolation, food Safe analysis and automated bead-based separations, including magnetic bead-based automated separations. In some embodiments, bioprocessing systems can include the use of labeled molecules, where the labels include, for example, immunofluorescent or fluorescent labels, including Qdot Nanocrystals or Alexa Fluor dye. In some embodiments, the protocol for processing a biomolecule is a protocol for processing a biomolecule immobilized on a solid support, such as a biomolecule-bound imprinted membrane. Likewise, the protocol may be a protocol for processing a Western blot (ie, immunoblot), Northern blot (Northern blot) or Southern blot (Southern blot). Once a protocol is initiated on a bioprocessing device, the automated bioprocessing systems, automated bioprocessing devices, automated bioprocessing cassettes, and automated bioprocessing methods disclosed herein provide automated "hands-off" bioprocessing while exhibiting performance that is: Similar manual handling is good, at least as good; minimizes contamination/cleanup; and/or increases efficiency and flexibility.

I. Biological treatment unit

In some embodiments, automated bioprocessing devices provided herein include automated devices that implement one or more protocols for processing biomolecules. In some embodiments, the bioprocessing device can be configured to run protocols and bioprocessing procedures selected from the group consisting of: immunoprecipitation, chromatin immunoprecipitation, recombinant protein isolation, nucleic acid isolation and isolation, protein labeling, isolation and isolation , cell separation and isolation, food safety, and automated bead-based isolation, including magnetic bead-based automated isolation.

In some embodiments, the automated bioprocessing apparatus includes one or more tanks for accommodating bioprocessing cartridges, such as two or more, three or more, four or more, five or more, or six one or more slots. Each tank may be individually configured to house and/or support a bioprocessing cartridge in the device and fluidly connect the cartridge with one or more fluid containers that may be used in the device. Each slot may also include a cartridge holder. In some embodiments, the tanks each include a fluid manifold connecting the bioprocessing cartridge to one or more process fluid supplies. In some embodiments, the slot includes an opening for receiving a cartridge and may guide one or more suction/extraction tubes into one or more process fluid containers, such as One or more process fluid containers placed in or included in the device. In some embodiments, each slot includes a single opening that includes at least one guide member, such as one or more suction/withdrawal tubes on one or both sides of the slot and/or cartridge holder. Projections or grooves in one or more process fluid containers, such as one or more process fluid containers placed in or included in the device fluid container. In some embodiments, the suction tube/extraction tube is a fluid connector necessary for the integral formation of the bioprocessing cartridge or can be connected to the bioprocessing cartridge. Each suction/withdrawal tube may be sized to perform a function identified according to a biological treatment protocol included in the central processing element of the device or according to a user-selected protocol. The lengths of the suction/extraction tubes may be the same as each other or may vary.

In some embodiments, each tank connects one or more control fluid supplies to a bioprocessing cartridge disposed in the tank. Such connections may include, for example, connections of control fluid manifolds to cartridges inserted into each slot. In some embodiments, the control fluid manifold is configured to form a sealed connection with a control fluid connector on a bioprocessing cartridge in the tank. In some embodiments, the control fluid manifold is connected or sealed to the bioprocessing cartridge in part by using a gasket or O-ring or other suitable sealing mechanism. The control fluid manifold may include individual supply connectors for interacting with each control fluid connector on the bioprocessing cartridge. In some embodiments, the control fluid manifold is reconfigurable, removable and/or replaceable, thereby providing alternative configurations for the control fluid connectors on the bioprocessing cartridge. In some embodiments, a pressurizable, inflatable, elastic container, such as a bag or bladder, is used which, when inflated, causes the supply connector to move toward the control fluid connector on the bioprocessing cartridge and to a connection such as sealingly connected to a control fluid connector on the bioprocessing cartridge such that the control fluid manifold can be pushed or connected to the control fluid connector on the bioprocessing cartridge. In some embodiments, the control fluid manifold can be connected to the control fluid connector using a mechanism such as a spring loaded mechanism or an automatic or manual locking mechanism.

In some embodiments, a control fluid manifold is used to provide control fluid, such as air pressure, vacuum, or pressurized fluid, to individual control channels on the bioprocessing cartridge through control fluid connectors that are fluidly connected to individual supply connectors. In some embodiments, a control fluid is used to provide pressure and/or vacuum to the non-contacting side (i.e., the side of the membrane not in contact with the process fluid) of a pump or valve membrane on the bioprocessing cartridge to activate the pump or valve. This actuation can be used to open or close a valve and/or to cause a pump to pump fluid through a process fluid channel on the bioprocessing cartridge. By using specifically defined schemes included in the automated control system, the pumps and valves can be controlled by supplying pressure and/or vacuum to said pumps, thereby controlling the actuation of the various valves or pumps in a specific sequence or according to specific instructions , in order to implement one or more biological treatments in the bioprocessing box.

Although the control fluids provided throughout the remainder of this application will be specifically referred to as air pressure and vacuum, it should be understood that other control fluids may be used to perform individual control steps, including other gaseous control fluids and other liquid control fluids, the gaseous control fluids Fluids such as nitrogen or oxygen or enriched air, etc. Additionally, in some embodiments, one or more control fluids may be treated prior to entering the bioprocessing cartridge and/or control fluid manifold. Such processing may include, such as purification by filtration, enrichment, pressure control, dehumidification, humidification, etc. to facilitate efficient processing.

Each of the individual supply connectors may be in fluid communication with a pressure source and/or a vacuum source. The pressure source and/or vacuum source may include an external pressure or vacuum source, such as a "house" air pressure supply ("air pressure supply") and/or a "house" vacuum supply ("house" vacuum supply). In some embodiments, the apparatus includes a vacuum pump and/or an air compressor, and one or both of said vacuum and air pressure are provided to the bioprocessing cartridge without the use of an external supply. The device may also include appropriate pressure gauges and regulators to help control the amount of pressure provided to each supply connector, thus controlling the channels and valves and pumps, and in some embodiments, the device includes a reservoir , which is used to store pressure and vacuum to supply to the cassette and to limit or avoid pressure or vacuum deficiencies or delays caused by pressure or vacuum supply delays, such as delays caused by the response time of the compressor and/or vacuum pump hour.

Alternatively, in some embodiments, rather than a control fluid manifold, some or all of the control fluid connectors on the bioprocessing cartridge may be individually connected to vacuum and pressure sources.

In some embodiments, the wells on the bioprocessing device can be reconfigurable, removable, and/or replaceable to provide different configurations, sizes, or types of bioprocessing cartridges. The tank may include various guide members, holes, retainers or any combination thereof to ensure that the bioprocessing cartridge remains in the tank at the correct height and orientation to perform the desired bioprocessing. In some embodiments, the cassette may be oriented vertically so that process fluid is introduced into the cassette in a generally upward direction and exits the cassette in a generally downward direction. This vertical orientation can be used to maintain space and can aid in efficient fluid removal from one or more bioprocessing chambers. In some embodiments, a cartridge may have one or more process fluid supplies that enter and/or exit the bioprocessing cartridge in a generally non-vertical orientation. In some embodiments, the cassette can be oriented horizontally so that fluid is pumped into and out of the cassette in a horizontal orientation. The fluid may be contained to prevent loss or spillage of the fluid, for example, by containing the fluid in a closed volume container that is deformable to expel the fluid from the container or to accept entry into the container. Fluid can be pumped into the bioprocessing chamber of the cartridge and then drawn out of the bioprocessing chamber of the cartridge using suction or vacuum pressure. The fluids may be positioned at the same level or at a level parallel to the cell or cell plane. In some embodiments, fluids and fluid containers can be oriented in a perpendicular orientation to a cassette or cassettes by connecting a vertical fluid container to a horizontal cassette or cassettes. The tubing of the cartridge is in fluid communication with the fluid container.

In some embodiments, the apparatus may include a removable fluid container tray comprising one or more sets of fluid container holders capable of holding fluid containers used during bioprocessing. The number of sets of fluid container holders may be any suitable number according to the protocol in which the containers are used. In some embodiments, the number of sets of fluid container holders is equal to or less than the number of slots in the device. In some embodiments, the number of sets of fluid container holders may be greater than the number of slots in the device. In some embodiments, such as those when fewer bioprocessing cartridges are used than the number of slots in the machine, empty fluid container holders may be provided below the empty slots.

The fluid container holders may be configured to accommodate waste containers, reagent containers and/or sample containers in a suitable size and number according to the biological process to be performed. For example, in some embodiments, a fluid container holder is provided that houses a set of fluid containers, where the set includes at least one sample container, at least one reagent container, and/or at least one waste container. In some embodiments, the fluid container holders may be configured to each accommodate different sized containers, depending on their function. For example, in some embodiments, the sample container is sized differently from one or more reagent containers, the waste container can also be sized differently from the reagent container or the sample container, or both, and the reagent containers can each be the same size of. In this manner, it should be understood that a set of fluid container holders may include a plurality of different sized holders for different sized containers. In some embodiments, the fluid container holder set is individually prepackaged with empty or prefilled reagent containers and available waste containers, and the fluid container holder set can be placed in a removable fluid container tray for operation The staff adds the sample containers to the appropriate available fluid container holders according to the predetermined protocol. In some embodiments, sets of fluid container holders are prepackaged with empty or prefilled reagent containers and usable waste containers to assemble a removable fluid container tray, or the sets of fluid containers The holder can be provided with a removable fluid container tray. In some embodiments, the fluid container holder is configured to share one or more containers. For example, fluid container holders may be configured as shared waste or reagent containers. In some embodiments, the fluid container holder and/or the removable fluid container tray are reusable or single use.

In some embodiments, the bioprocessing device further includes an automated control system, including a computerized control system configured to control at least one parameter associated with a bioprocessing procedure or protocol. In some embodiments, the computer control system may be configured to control one or more of the following non-limiting examples: actuation of one or more inlet valves located on the bioprocessing cartridge; activation of one or more inlet valves located on the bioprocessing cartridge; Activation of one or more pumps; amount of one or more process fluids provided to one or more bioprocessing cartridges; mixing of one or more process fluids; one or more solid supports versus one or more Exposure time of one or more process fluids, pumping and flow paths of one or more process fluids, circulation of one or more process fluids; pumping flow rates, sequences, pump delays, and addition from one or more bioprocessing cartridges and/or purge the volume of process fluid and provide pressure and/or vacuum to one or more bioprocessing cartridges, such as to one or more control fluid channels on the bioprocessing cartridge.

In some embodiments, the automated control system includes one or more of: a computerized control system including a central processing element, a graphical user interface ("GUI"), and an operator input system. The central processing element may include memory and one or more microcontrollers. In operation, a user may use the operator input system through the GUI and software or firmware loaded on the computer control system to select one or more stored protocols or to enter one or more user-created protocols that indicate a One or more microcontrollers opens a series of valves and pumps on one or more bioprocessing cartridges in one or more tanks within the apparatus. Valve and pump activation can be controlled with pressure and vacuum supplies. These valves and pump activations can be used to pump process fluids, including reagents and samples, from containers in fluid container holders into the process fluid pathways and bioprocessing chambers of bioprocessing cartridges, and to pump process fluids, including reagents and samples Process fluid is pumped from the bioprocessing cartridge into one or more fluid containers and samples are processed according to the selected protocol. In some embodiments, using the same or different types of fluid container holder sets, containers, reagents, and samples per protocol, the automated control system can implement the same or different bioprocessing cartridges on multiple bioprocessing cartridges of the device. plan. During processing, the GUI may provide the user with the ability to view the progress of one or more protocols ongoing on the bioprocessing cartridge, and the computer control system may provide alerts indicating processing completion or errors or other problems that may occur during processing. In some embodiments, the bioprocessing unit and/or the computer control system may also include a safety interlock that shuts down the system if the unit is in one or more unsafe or unready states. Preventing a run protocol, such as, as non-limiting examples, container trays not fully inserted into the apparatus, one or more bioprocessing cartridges not inserted properly into slots or not properly connected to air and/or vacuum supplies, air or vacuum supplies Insufficient, air or vacuum supply exceeds safe limits and/or electrical supply is insufficient or exceeds safe limits.

The operator input system may include any suitable input system such as a keyboard, keys, mouse, touch screen, or any other suitable device used by a user to interface with the computer system and to run software or firmware. The software or firmware used in the bioprocessing device may be proprietary or may be commercial off-the-shelf software. In some embodiments, a bioprocessing device may include sensors for monitoring the progress of one or more protocols running on the device. For example, the device may include pressure and vacuum sensors, flow rate sensors, temperature sensors, time lag sensors for detecting the pressure and vacuum provided during each step of the protocol, detecting the relationship between one or more processing steps performed on the device Associate any parameter with the sensor. Sensors may provide passive sensing of various parameters recorded in the control system or may provide active sensing for controlling processes and guiding processes. Such control can occur using any suitable control procedure, including, but not limited to, proportional, integral, proportional-integral or proportional-integral-derived control.

In some embodiments, the computer control system also includes means for remotely controlling the device and/or for uploading information, such as software or firmware updates, into the computer control system and for downloading and/or storing and implementing on the device. One or more means and/or mechanisms for operating the associated information. For example, via a direct connection such as an Ethernet interface, a Personal Computer Memory Card International Association (PCMCIA) slot, or a Universal Serial Bus (USB) interface, via a wireless connection, via a device such as a flash drive or thumb drive , a portable storage medium writable on CD-ROM or DVD, the device can upload information to the device or download any processing parameters used for operation directly to the computer, or can connect the device to a network such as a LAN or WAN, Or connect to Internet-based applications. In addition, the device can provide a secure record of process parameters, thereby preventing or ensuring that the actual operating parameters have not changed after a run has been performed; and can provide date/time confirmation of the run. In some embodiments, the software or firmware is configured to interact with an electronic notebook program, such as a secure electronic notebook program, to download processing parameters and operating data directly to the program.

In some embodiments, the device is fabricated at a size that allows use on a typical laboratory table or in a chemical fume hood, and at various temperatures such as room temperature or in a cold room.

In use, the automated control system verifies that the bioprocessing cartridge is correctly inserted into the device and can then accept user-selected protocols (pre-loaded or user-created). The bioprocessing device may be preprogrammed with a protocol such as a western protocol or a nucleic acid purification protocol or a southern protocol. In some embodiments, the bioprocessing device can store other protocols. The device may allow a user to select from a list of existing protocols or to create and store a user-defined protocol, eg, a user-defined western or nucleic acid purification protocol. In some embodiments, the device may also allow for more consecutive, reproducible run-to-run experiments. The device can also be programmed with protocols that do not require run-to-run re-optimization. After the protocol is selected, the automated control system will activate the valves and pumps according to the selected protocol to pump the fluid from the fluid container into the bioprocessing cartridge and the bioprocessing chamber therein, so as to implement the desired biological treatment on the cartridge.

II. Bioprocessing box

In some embodiments, a bioprocessing cartridge can include a mesofluidic circuit or a microfluidic circuit formed from one or more substrate layers. In some embodiments, a bioprocessing cartridge may include one or more additional layers or elements between one or more substrate layers. In some embodiments including one or more other layers or elements, at least one other layer or element may include one or Multiple films or layers.

In some embodiments, a bioprocessing cartridge can include at least one bioprocessing chamber configured to house a solid support and a plurality of mesoscale and/or microscale bioprocessing chambers in direct or indirect fluid communication with the bioprocessing chamber via at least one pump. Fluid flow channels, such as pumps integral with or included in or on the cassette or layer(s) making up the cassette, as necessary. In some embodiments, the fluid flow channels may include process fluid channels and/or control fluid channels. In some embodiments, a bioprocessing cartridge can include two or more fluidic layers. In some embodiments, a bioprocessing cartridge may have at least one process fluid layer and a control fluid layer. In some embodiments, there may be direct or indirect communication between the process fluid layer and the control fluid layer. In some embodiments, the process fluid layer may include a layer having a plurality of process fluid channels thereon, and the control fluid layer may include a layer having a plurality of control fluid channels therein.

As used herein, the term "microscale" means that at least one cross-sectional dimension of a flow channel or other structural element is on the order of about 0.1 μm to about less than 1000 μm, such as about 0.1 μm to about 500 μm, about 10 μm to about 250 μm Or about 100 μm to about 250 μm, the term “mesoscale” means that at least one cross-sectional dimension of a flow channel or other structural element is on the order of about 1000 μm to about 4 mm, such as about 1000 μm to about 3.5 mm, about 1000 μm to about 2.5 mm , about 1000 μm to about 1.5 mm, or about greater than 1000 μm, about greater than 1100 μm, about greater than 1250 μm, or about greater than 1500 μm.

In some embodiments, a bioprocessing cartridge can include a plurality of mesoscale and/or microscale process fluid flow channels. In some embodiments, a process fluid flow channel can provide a fluid connection between a process fluid connector on a bioprocessing cartridge and one or more pumps, inlet valves, and/or process valves on the bioprocessing cartridge. In some embodiments, process fluid flow channels may be used to provide process fluid, such as reagents and/or samples, through various valves and pumps on the bioprocessing cartridge, and into any one or more of the bioprocessing cavity. In general, process fluid channels may direct any process fluid, such as reagents and/or samples used in the actual processing performed by the bioprocessing cartridge, into the right place at the right time. In some embodiments, one or more process fluid flow channels may be provided on a different fluid layer than the control fluid flow channels.

In some embodiments, a bioprocessing cartridge can include a plurality of mesoscale and/or microscale control fluid flow channels. In some embodiments, the control fluid flow channels are microscale. In some embodiments, the control fluid flow channel is mesoscale. In some embodiments, a control fluid flow channel may provide a fluid connection between a control fluid connector on a bioprocessing cartridge and one or more pumps, inlet valves, and/or process valves on the bioprocessing cartridge. In some embodiments, the control fluid flow channels can be used to provide pressure or vacuum to the pump membranes and valve membranes on the bioprocessing cartridge alone to activate the pumps or open or close the valves. In some embodiments, the control fluid flow channels may be on a different fluidic layer of the bioprocessing cartridge than the process fluid channels.

In some embodiments, the bioprocessing cartridge may be a disposable cartridge. Disposable cartridges reduce the risk of cross-contamination between runs. The cartridge is configured in such a way that successive quantities of solutions or reagents can be delivered to the cartridge in a manner that increases reproducibility.

In some embodiments, a control fluid connector can be configured to connect a plurality of control fluid flow channels to an automated control system. In some embodiments, each control fluid flow channel can be in fluid communication with a separate control fluid connector. In some embodiments, the control fluid connector can be configured to communicate with an automated control system through a control fluid manifold. In some embodiments, a bioprocessing cartridge can be configured to be received in a cartridge holder of a bioprocessing device that causes a control fluid connector to be in fluid communication with a supply connector on the bioprocessing device that is connected to a control fluid The manifolds are in fluid communication. In some embodiments, using an expandable bladder or bag, a mechanical or manual latching mechanism or an electrical latching or connection mechanism, or any other suitable mechanism for facilitating fluid communication between the control fluid connector and the supply connector, The urging can be done.

In some embodiments, a bioprocessing cartridge may include one or more bioprocessing chambers including, but not limited to, one bioprocessing chamber, two bioprocessing chambers, two or more bioprocessing chambers, three bioprocessing chambers , three or more bioprocessing chambers, four bioprocessing chambers, four or more bioprocessing chambers, five bioprocessing chambers, or five or more bioprocessing chambers, or any suitable number of bioprocessing chambers. For example, the bioprocessing chamber can be closed or sealed by any suitable sealing means, including the use of gaskets, o-rings, welding, clips, etc., or in some embodiments, an operator or user has access to a or multiple bioprocessing chambers. In some embodiments, the bioprocessing chamber can have inlets and outlets on different fluidic layers of the bioprocessing cartridge, while in other embodiments the bioprocessing chamber can have inlets and outlets on the same fluidic layer. In some embodiments, the volume of the processing chamber should be suitable to allow fluid to flow freely through the solid support therethrough or over the surface of the solid support therethrough. In some embodiments, the bioprocessing chamber has a fluid volume of about 1 μl to about 100 ml, such as about 10 μl to about 100 ml, about 20 μl to about 100 ml, about 50 μl to about 100 ml, about 100μl-about 100ml, about 150μl-about 100ml, about 200μl-about 100ml, about 250μl-about 100ml, about 300μl-about 100ml, about 500μl-about 100ml, about 750μl-about 100ml, about 1ml-about 100ml, about 2ml- About 75ml, about 3ml-about 60ml, about 4ml-about 50ml, about 4ml-about 45ml, about 4ml-about 40ml, about 4ml-about 35ml, about 5ml-about 30ml, about 10ml-about 25ml or about 15ml-about 20ml .

A bioprocessing chamber may include a fluid mixing chamber and/or may house a solid support for processing one or more samples. A solid support can be any support used for filtering, washing, staining, eluting, collecting, processing, or performing chemical reactions or biological treatments. In some embodiments, the solid support can be selected from one or more of the following: filter boxes, filter papers, precipitation membranes, precipitation filters, solid phase extraction columns, solid phase extraction boxes, solid phase extraction discs, Resins, membranes such as blotting membranes, filter membranes, PVDF membranes, nylon membranes, positively charged nylon membranes, and nitrocellulose membranes, reaction beads such as glass beads and magnetic beads, arrays containing biomolecules such as nucleic acid microarrays, protein arrays Rigid planar solid supports for tissue arrays, microscope slides and combinations thereof.

In some embodiments, the solid support located in one or more bioprocessing chambers can include filter paper, filters, or filter cartridges. The filter paper, filter or filter cartridge may comprise suitable chemical properties, pore size, shape and three-dimensional structure and for the intended use and may be configured for longitudinal flow (flow through), cross flow (cross flow), cut Any suitable type of filter of surface area to tangential flow or any combination thereof, the three-dimensional structure such as symmetrical or asymmetrical three-dimensional structure including "V" shaped or funnel shaped pore structures. Filter paper, filters or cartridges may comprise any suitable material such as polyphenylene ether sulfone, polyethylene, ultra-high molecular weight polyethylene, polypropylene, nylon, cellulose, cellulose triacetate, polyacrylonitrile, polyamide, fiberglass , silica, polysulfone, PVDF, etc. In some embodiments, the solid support located in one or more biological processing chambers may include a sedimentation membrane or sedimentation filter. In some embodiments, the solid support located in one or more bioprocessing chambers may comprise a solid phase extraction cartridge, solid phase extraction cartridge, or solid phase extraction disc. In some embodiments, the solid support located in one or more bioprocessing chambers can comprise an imprinted membrane. Filters, filter papers or filter cassettes can be layer or multi-layer filters. In some embodiments, the solid support located in one or more bioprocessing chambers can include a variety of beads, such as coated beads, coated glass beads, glass beads, magnetic beads, or coated magnetic beads.

In some embodiments, the bioprocessing chamber is open and provides access for the user to insert the solid support. In some embodiments, the bioprocessing chamber is open and configured to accommodate a blotting membrane with or without a blotting membrane holder. The blotting membrane retainer can be used to prevent the blotting membrane from contacting or sticking to a surface of the bioprocessing chamber, and can be used to facilitate the flow of various process fluids around and through the blotting membrane surface. Additionally, in some embodiments, the bioprocessing chamber may include additional space to accommodate foam formed during processing of the sample. For example, in some embodiments, if a bioprocessing chamber is open and a blotting membrane with or without a blotting membrane holder is inserted by the user, the bioprocessing chamber can be sized to The top of the chamber provides additional space to prevent overflow of foam formed during bioprocessing. Additionally, the top of the bioprocessing chamber may be configured such that the top of the chamber is wider than the bottom. This increased width of the top provides additional volume to accommodate foam formation, if present.

In some embodiments, a bioprocessing cartridge can include one or more flow control elements. In some embodiments, the one or more flow control elements may be selected from pumps, process valves, check valves, inlet valves, layer pass-throughs, and/or pass-through valves. In some embodiments, one or more flow control elements can be placed in a flow path defined by one or more of a plurality of fluid flow channels on a bioprocessing cartridge.

In some embodiments, the bioprocessing cartridge can include about 1 to about 10 pumps, such as about 1 to about 8 pumps, about 1 to about 6 pumps, about 1 to about 5 pumps, or about 1 to about 4 pumps . The pump may be included in or on the bioprocessing cassette or at least one layer of the bioprocessing cassette, or the pump may be required for the integral formation of the bioprocessing cassette or at least one layer of the bioprocessing cassette. The pump may be used to pump process fluid from a process fluid container into or out of the cartridge and through a plurality of process fluid channels on the cartridge. In some embodiments, applying pressure and/or vacuum to the pump membrane may actuate the pump using a control fluid channel associated with the pump on a different fluid layer than the process fluid channel associated with the pump. In this way, the pump may comprise a membrane pump similar to a diaphragm pump. In some embodiments, the pump, when activated, can cause turbulence in the fluid flow in the treatment channel, while in other embodiments, the fluid flow in the channel can be laminar or transitional.

In some embodiments, the bioprocessing cartridge may include about 1 to about 20 inlet valves, such as about 2 to about 18, about 3 to about 16, about 4 to about 14, about 5 to about 13, about 6 - about 12 or about 7 - about 10 inlet valves. In some embodiments, an inlet valve controls the entry and exit of process fluid into and out of the bioprocessing cartridge, and is typically in direct fluid communication with one or more process fluid connectors on the bioprocessing cartridge. In some embodiments, using a control fluid channel associated with the inlet valve that is on a different fluid layer than the process fluid channel associated with the valve, applying pressure or vacuum to the inlet valve membrane can actuate the inlet valve thereby Opening or closing, thereby opening or closing, fluid communication between at least one process fluid channel on the bioprocessing cartridge and a process fluid connector on the bioprocessing cartridge. When closed, the inlet valve can prevent flow of process fluid into or out of the bioprocessing cartridge. In some embodiments, the bioprocessing cartridge can include an inlet valve in each flow path between the process fluid connector and each of the plurality of fluid flow paths. In some embodiments, at least two of the plurality of process fluid connectors share an inlet valve.

In some embodiments, a bioprocessing cartridge may include about 1 to about 25 process valves, such as about 1 to about 22, about 1 to about 20, about 1 to about 18, about 2 to about 16, about 2 - about 14, about 3 to about 12, about 3 to about 10 or about 3 to about 8 process valves. Depending on the individual process, the process valve can be actuated to open or actuated to close. In some embodiments, the process valve may be actuated by applying a supplied pressure or vacuum to the process valve membrane through a control fluid channel on a different fluid layer than the process fluid channel associated with the process valve. . After fluid enters the bioprocessing cartridge through one or more inlet valves, the process valve can be used to direct the fluid to the appropriate location on the bioprocessing cartridge at the appropriate time, according to protocol. In some embodiments, the bioprocessing cartridge includes at least one process valve located in the flow path between the pump and the bioprocessing chamber.

In some embodiments, a bioprocessing cartridge can include a check valve. In some embodiments, a bioprocessing cartridge can include at least one check valve. In some embodiments, a check valve or valves may allow flow through the valve in only one direction, including flow between layers, and through pressure provided to the valve membrane via a control fluid channel or through a flow channel. Process fluid flowing in the correct direction is activated by the associated pressure, thereby opening for flow in that direction. In some embodiments, check valves are provided to supply control fluid between two different layers of the bioprocessing cartridge. In some embodiments, the control fluid may be provided as a process fluid, such as to provide air pressure to dry filters or membranes or to drain residual fluid from pumps, channels, or bioprocessing chambers. In some embodiments, the check valves may allow fluid flow through them in one direction, and between layers in one direction. In some embodiments, check valves are provided that allow flow through them in either direction, but allow flow through them in only one direction, such as from the process fluid layer to the control fluid layer of the bioprocessing cartridge or from The control fluid layer to the process fluid layer of the bioprocessing cartridge. In some embodiments, the bioprocessing cartridge includes at least one inspection valve that is a process valve or an inlet valve.

In some embodiments, a bioprocessing cartridge can include at least one layer channel or channel valve. Layer channels or channel valves can provide flow of process fluid between different fluid layers of the bioprocessing cartridge. For example, if the bioprocessing chamber inlet is on a different fluid layer than a given flow channel associated with the fluid for the chamber, the flow channel may direct fluid to a layer channel or channel valve , where fluid is transferred from one fluid layer to the fluid layer associated with the process chamber inlet, and then continues into the process chamber through process fluid channels on the other fluid layer. Layer channels are usually passive fluid connections between fluid layers, while channel valves can be activated by pressure in the fluid channel itself or by controlling the fluid, opening or closing as needed to control the flow of fluid from one layer to another.

In some embodiments, a bioprocessing cartridge includes one or more process fluid connectors. In some embodiments, the process fluid connectors may include tubes or tube-like structures configured to fluidly connect the cartridge to one or more external (ie, not on the bioprocessing cartridge) fluid containers. Fluids can be introduced into or removed from the cartridge and into or out of one or more fluid containers through process fluid connectors and then transported through fluid flow channels into various parts of the bioprocessing cartridge . In some embodiments, a bioprocessing cassette or cassettes may include about 3 to about 20 process fluid connectors, such as about 4 to about 18, about 5 to about 16, about 6 to about 14, or about 8 - about 12 process fluid connectors or about 3 or more process fluid connectors, about 4 or more process fluid connectors, about 5 or more process fluid connectors or about 6 or more process fluid connectors Connector. In some embodiments, the process fluid connector is connected to a process fluid manifold on the biological processing device through which various process fluids can be provided to the biological process fluid through one or more of a plurality of flow channels. Process cartridges and remove from the bioprocessing cartridges. In some embodiments, each process fluid connector holds fluid from a separate fluid container, where each container may hold the same or a different fluid than the other container.

In some embodiments, the process fluid connector may be in fluid communication with the process fluid reservoir via a suction/extraction tube, which may be in fluid communication with the process fluid connector or the process fluid connector may be a suction/extraction tube Withdraw the tube. When placed in the tank of a bioprocessing device, the suction/withdrawal tube can protrude from the cassette and into the fluid reservoir. In one embodiment, when the cartridge is inserted into the cartridge well, the suction/withdrawal tube may extend into a reservoir located below the cartridge well. The reservoir can be any tube, bottle, vial or similar reservoir capable of holding fluid. In some embodiments, the reservoir may include a seal that may allow the tip of the suction/withdrawal tube to enter the reservoir, but also reduce the risk of foreign matter entering the reservoir, or that may reduce the risk of storage Loss of reagent or fluid in cell. The suction/extraction tubes may be the same length or different lengths from each other, depending on the size and/or shape of the reservoir or reservoirs. While the suction/extraction tube may be used to carry process fluid from the reservoir into the cassette, the suction/extraction tube may also be used to drain fluid as waste from the cassette into one or more storage reservoirs.

Additionally, in some embodiments, in conjunction with the suction/extraction tube and the bioprocessing cassette or cassettes, the container can be used to hold one or more containers or mixing containers for process fluids during bioprocessing. For example, in some embodiments, fluid may be moved from one process fluid container into a bioprocessing cartridge, then removed from the bioprocessing cartridge, and into a different container. In this way, with the bioprocessing cartridge providing mixing of fluids, fluids can be pumped back and forth between containers. Alternatively, in some embodiments, fluid can be removed from and returned to the same container, providing mixing of the fluids without combining it with any other fluid. However, in some embodiments, one or more containers and a container holder or holders, container trays, and bioprocessing devices can be configured to provide agitation of fluids in the containers, for example, magnetic stir bars or for Any other suitable agitation mechanism that stirs or agitates the fluid in the container.

In some embodiments, a bioprocessing cartridge can include one or more preloaded or loaded process fluids or processing reagents. In such embodiments, the process fluid or treatment reagent may be included in a separate reservoir or chamber of the cartridge, or may be included in one or more pumps or channels of the cartridge. In some embodiments, a preloaded or loaded process fluid or treatment reagent may include a process fluid or a component of a process fluid. In some embodiments, a preloaded or loaded process fluid or treatment reagent may include a treatment reagent that dissolves in the process fluid after exposure to the process fluid.

In some embodiments, bioprocessing cartridges can be designed to be reusable. In some embodiments, the cartridge can be disposable and/or can be designed for a specific or limited number of uses, such as about 20 uses or less, about 15 uses or less, about 10 uses or more Few, about 9 uses or less, about 7 uses or less, about 5 uses or less, or about 3 uses or less. In some embodiments, protocols can be provided on the automated control system to provide for cleaning of reusable cartridges prior to reuse. Thus, in some embodiments, the cartridge may be a consumable item.

In some embodiments, the bioprocessing cartridge may be a single-use cartridge. In some embodiments, the cartridge may include an indicator configured to signal when the cartridge has been used and/or has not been used. In some embodiments, the indicator can be any suitable indicator for indicating when the cartridge has been used and/or has not been used. In some embodiments, the indicator can be a color indicator located in a suitable location on the card, such as in a color indicator cavity, which can change color continuously during bioprocessing to indicate that the cartridge is in use pass. In some embodiments, the automated control system of the bioprocessing device can be configured to detect a color change and prevent operation when a spent card is placed in the slot. In some embodiments, the automated control system of the bioprocessing device can be configured to detect the indicator color before and after the color change, and prevent or stop operation when one or both colors are not observed at different times .

In some embodiments, the indicator may comprise an absorbent substrate such as a white absorbent substrate, such as a paper strip impregnated with red dye on one side and white on the other, so that when the substrate is wetted, the dye diffuses throughout the The matrix, which turns the white side to red. In some embodiments, the white side is initially presented to a detector of the device. When the cartridge is used, then the detector can detect a white to red change in the indicator. In some embodiments, the color change can be induced by directing some of the fluids used in the bioprocessing device into separate chambers containing a strip of color indicator tape. The tape can remain wetted by including fluid that is allowed to flow into the tape chamber but prevented from returning to the process fluid channel after it has interacted with the dye. Furthermore, once the indicator material is wetted, the dye redistributes throughout the matrix, turning the white side to red, and the color remains in the matrix even after the matrix dries. Examples of suitable color indicating tapes are described in US Patent No. 7,105,225, which is incorporated by reference in its entirety. Additionally, suitable indicator tapes may include water contact indicator tape, such as 3M Water Exposure Indicator Tape 5557, 5558 or 5559.

In some embodiments, the water-sensitive paper or tape may comprise a small circular piece of paper or tape (approximately 5-15 mm, such as 8 mm in diameter) placed between two layers of the bioprocessing cartridge and within the cavity, when the card is removed from the After removal from the instrument, the cavity allows the customer to clearly see the small circular piece of paper or strip, and when the cartridge is inserted into the instrument, the cavity allows the electronic color sensor to clearly detect the Small piece of circular paper tape or strip. When the cartridge is used during operation, a small portion of the first fluid in the cartridge enters the cavity containing the paper or tape, causing a color change. This change can occur after less than about 20 seconds of runtime. An electronic color sensor, such as a color sensor integrated with colored LED lights, can be embedded in a bioprocessing device, such as a cartridge well. When running a program, after the user presses the "Run" key, the color sensor detects the color of the paper or tape and can send the information to the GUI, if the wrong color is detected, the GUI will prompt the user to insert a new card . In some embodiments, the system may also allow for second color detection during operation. In some embodiments, the second color detection is performed as follows: after running for a set length of time, such as about 1 minute to about 10 minutes, for example about 2 minutes or more, about 3 minutes or more, For about 4 minutes or more or about 5 minutes or more, the GUI interrogates the sensor again to determine if the color of the paper or tape has changed. If the color of the paper or tape does not change, display an error message and stop the protocol.

In some embodiments, a color sensor can determine a color change as follows. When calibrated, each sensor can output a signal in response to a standard red card and a standard white card. The midpoint of those signals (kHz) is determined as the dividing point between what is considered red (used) and what is considered white (unused). This information can be stored permanently in the instrument memory. In user operation, this information is used for the first color detection described above. For the second check, after a set time, another check is performed, say after running for 2 minutes, and the returned value is compared with the first. If 1kHz changes to 1 or more, it is determined that the card is not disturbed and the operation continues. If no changes are detected, the run can be terminated and the user instructed to insert a new cartridge or cartridges.

In some embodiments, a color sensor can determine a color change as follows. During a calibration period, a series of measurements are taken at set intervals to establish a baseline color level. After a calibration period, color measurements are produced at set time points. Each measurement is compared to baseline levels. Once the detected color level is greater than about 2 standard deviations, greater than about 3 standard deviations, greater than about 4 standard deviations, or greater than about 5 standard deviations from the baseline color level, the color is considered to have changed and the card is considered not to be disturbed, Run continues. If the color change as measured by the appropriate number of standard deviations does not occur, the run may be terminated and the user instructed to insert a new cartridge or cartridges.

In some embodiments, a bioprocessing cartridge may include one or more pumps, valves, process fluid channels, and/or control fluid channels as required for the integral composition of the cartridge, such as included on one or more layers of the cartridge. In some embodiments, the process fluid channel and/or the control fluid channel are rigid flow channels or have one or more rigid walls. The channels may have any suitable cross-sectional shape, including circular, oval, square, rectangular, D-shaped, and any other suitable polygonal cross-section. In some embodiments, one or more channels may have a D-shaped cross-section or a square or rectangular cross-section, wherein a substantially semicircular portion of the channel (for a D-shaped cross-section) or three sides of the channel (for square or rectangular cross-section) with rigid walls, and wherein the flat part (D-shape) of the channel or the fourth side of the channel is formed by a thin film, which may be the same or different material as the rigid wall, And it is flexible or less rigid or harder than the material used for the rigid walls. In some embodiments, the flexible or less rigid material is flexible or less rigid due to thinness rather than due to inherent material properties of the flexible or less rigid material. For example, in some embodiments, one or more process fluid channels or control fluid channels may have a D-shaped or square or rectangular cross-section, wherein one or more of the channel's flat sides (D-shaped) or one of the sides (square or rectangular cross-section) comprising a metallic or plastic film or foil, while the other side or sides of one or more channels comprise plastic. In some embodiments, the process fluid channels and/or control fluid channels are not flexible tubes.

In some embodiments, the bioprocessing cartridge may be of conventional shape, such as rectangular, square or substantially rectangular or substantially square, while in other embodiments the bioprocessing cartridge may have a more complex polygonal shape or may be substantially polygonal shape or may be irregular in shape. In some embodiments, at least one dimension of the cassette is longer than about 5 cm, such as at least one dimension is from about 6 cm to about 40 cm, such as from about 7 cm to about 37 cm, from about 8 cm to about 35 cm, from about 9 cm to about 30 cm, from about 10 cm to about 25 cm, About 11 cm to about 22 cm, about 12 cm to about 21.5 cm, about 12 cm to about 20 cm, about 12 cm to about 18.5 cm, about 14 cm to about 18.5 cm, excluding any addition to the dimensions of the bioprocessing cartridge due to swelling, including but not limited to Bumps such as process fluid connectors, control fluid connectors, and suction/extraction tubing. In some embodiments, the bioprocessing cartridge can be substantially flat, having at least one dimension ("depth") substantially shorter than one or two of the other dimensions. In some embodiments, the depth of the bioprocessing cartridge is less than about 50% of the longest dimension measured above, such as less than about 40% of the longest dimension, less than about 30% of the longest dimension, less than about About 25% of the dimension, less than about 20% of the longest dimension, less than about 15% of the longest dimension, less than about 10% of the longest dimension, or less than about 7.5% of the longest dimension.

In some embodiments, a bioprocessing cartridge can include one or more cartridge alignment guides to ensure that the cartridge is inserted into the cartridge slot in the correct orientation. In some embodiments, the cartridge alignment guide may be a tab or tabs or a protrusion or protrusions or a slot or slots that are mounted on and into which the cartridge is inserted. in the opening of the corresponding cartridge slot.

III. Biological treatment methods

In some embodiments, the bioprocessing method comprises: providing a bioprocessing cartridge, wherein the cartridge includes at least one bioprocessing chamber containing a solid support and a plurality of mesoscale and/or microscopic and pumping at least one process fluid through at least one of the plurality of process fluid channels and into the at least one biological processing chamber. In some embodiments, the pumping includes pumping one or more process fluids and/or samples into the processing chamber and into contact with the solid support. The process fluid delivered to the cartridge solid support can be any suitable solvent, solution or reagent used in the desired biological processing, including but not limited to liquid reagents for chemical reactions, solvents or solutions for washing, Antibody solutions, buffers, blocking buffers, and solutions containing fluorescently labeled reagents. The process fluid may also include samples to be processed, such as proteins, nucleic acids and other macromolecules, cells, cell lysates, and combinations thereof. In some embodiments, at least one process fluid includes at least one blocking buffer. In some embodiments, at least one process fluid includes at least one antibody. In some embodiments, at least one process fluid includes at least one wash liquid. In some embodiments, the bioprocessing method can include pre-treating the cells prior to pumping. In some embodiments, pretreatment of cells may include adding salts, eg, NaCl, MgCl ₂ , CaCl ₂ , NH ₄ Cl, etc., to the cells. Cells may be pretreated to increase plasmid production by any suitable method of pretreating cells. In some embodiments, the concentration of the saline solution can be any suitable concentration for enhancing yield, for example, a concentration of about 0.1M to about 0.5M, about 0.2M to about 0.5M. In some embodiments, the salt concentration can be any suitable concentration for increasing plasmid yield by at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 65%.

In some embodiments, pumping at least one process fluid into at least one bioprocessing chamber through at least one of the plurality of channels includes pumping at least one process fluid through a flow channel proximate a bottom of the chamber. In some embodiments, pumping at least one process fluid into at least one bioprocessing chamber through at least one of the plurality of channels includes pumping at least one process fluid through a flow channel proximate a top of the chamber. In some embodiments, pumping at least one process fluid into at least one bioprocessing chamber through at least one of the plurality of channels includes pumping at least one process fluid through a flow channel proximate a side of the chamber.

In some embodiments, pumping at least one process fluid into at least one bioprocessing chamber through at least one of the plurality of channels includes passing at least one process fluid from the bioprocessing chamber through a fluid flow channel near the bottom of the chamber flow through and return to the chamber through a fluid flow channel near the upper portion of the chamber.

In some embodiments, pumping at least one process fluid into at least one bioprocessing chamber through at least one of the plurality of channels comprises passing at least one process fluid from the bioprocessing chamber through a fluid flow channel near the top of the chamber and return to the chamber through a fluid flow channel near the bottom upper portion of the chamber.

In some embodiments, pumping at least one process fluid into at least one bioprocessing chamber through at least one of the plurality of channels includes flowing at least one process fluid from the bioprocessing chamber through fluid flow proximate the side of the chamber The channel communicates and returns to the cavity through a fluid flow channel near the bottom of the cavity.

In some embodiments, pumping at least one process fluid through at least one of the plurality of channels into at least one bioprocessing chamber includes pumping at least one process fluid through a filter or membrane into at least one bioprocessing chamber. In some embodiments, the filter membrane comprises a blotted membrane. In some embodiments, the filter membrane comprises a western blot membrane. In some embodiments, the filter comprises a cell separation membrane or a cell capture membrane. In some embodiments, the filter comprises a lysate clarification filter. In some embodiments, the filter comprises a nucleic acid precipitation filter. In some embodiments, the filter comprises a nucleic acid purification filter. In some embodiments, at least one pore size of the one or more filters or one or more layers of filters is from about 0.2 μm to about 3 μm, from about 0.45 μm to about 2.5 μm, from about 0.5 μm to about 2.4 μm, from about 0.5 μm to about 2.4 μm, about 0.65 μm to about 2.2 μm, about 0.8 μm to about 2.0 μm, about 1.0 μm to about 1.5 μm, or about 1.2 μm to about 1.5 μm. In some embodiments, the filter membrane includes two layers of cell separation filters or capture filters, one layer having a pore size of about 1.0 μm to about 1.5 μm, such as about 1.2 μm, and one layer having a pore size of about 0.5 μm to about 1.5 μm. About 0.8 μm, such as about 0.65 μm. In some embodiments, the filter membrane may comprise a two-layer nucleic acid precipitation filter, one layer having a pore size of about 1.0 μm to about 1.5 μm, such as about 1.2 μm, and one layer having a pore size of about 0.5 μm to about 0.8 μm , such as about 0.65 μm. In some embodiments, filter membranes may include cell separation or capture filters or nucleic acid precipitation filters, which may include asymmetric filters with pore sizes ranging from about 0.65 μm to about 2 μm. In some embodiments, the filter may comprise a glass fiber lysate clarification filter having a pore size of about 0.8 μm to about 1.5 μm, such as about 1.0 μm.

In some embodiments, pumping at least one process fluid into at least one bioprocessing chamber through at least one of the plurality of channels includes adding a blocking buffer to the bioprocessing chamber, passing the blocking buffer across the surface of the blotting membrane and circulating to form a closed blotting membrane, adding at least one antibody solution to the bioprocessing chamber; and recirculating at least one antibody solution across the surface of the blocked blotting membrane. In some embodiments, recirculating the at least one antibody solution across the surface of the blocked blotting membrane may comprise, recirculating the primary antibody solution across the surface of the blocked blotting membrane; washing the blotting membrane; Add the secondary antibody solution; and recirculate the secondary antibody solution across the surface of the washed blot.

In some embodiments, the action of pumping can provide additional mixing in the bioprocessing chamber. For example, in some embodiments, if the process fluid is circulated or recirculated in a fluid flow channel near the top or sides of the chamber and returned to the chamber through a fluid flow channel near the bottom of the chamber, the same as The pressure associated with the pumping action into or back into the chamber may create localized eddies or swirl regions that promote flow of process fluid through the bioprocessing chamber and mixing of the process fluid in the chamber. In some embodiments, when this pumping method is used in the presence of a blotted membrane with or without a blotted membrane holder, the pumping action can be such that each pumping cycle deactivates the blotted membrane. The slight movement of the membrane in the bioprocessing chamber serves to ensure that all sides of the imprinted membrane are exposed to the process fluid in a substantially uniform manner and to prevent the imprinted membrane from sticking to the surface of the bioprocessing chamber or to the imprinted membrane The role of the retainer.

In some embodiments, pumping at least one process fluid through at least one of the plurality of channels into at least one bioprocessing chamber comprises pumping at least one process fluid through at least one solid phase extraction column, at least one solid phase The extraction membrane, at least one solid phase extraction cartridge or at least one solid phase extraction disc, enters the bioprocessing chamber. In some embodiments, the solid phase extraction column, solid phase extraction membrane, solid phase extraction cartridge or solid phase extraction disc comprises a silica reversible binding ligand or a charge-switch binding ligand.

In some embodiments, pumping at least one process fluid through at least one of the plurality of channels into at least one bioprocessing chamber comprises pumping cell culture medium or cells suspended in solution through a cell separation filter, To separate the cells from the cell culture medium, where the cells are captured on a filter. In some embodiments, the pumping can also include resuspending the captured cells in a lysate to form a lysate; neutralizing the lysate; and clarifying the lysate. The cells can be resuspended in a lysate and lysed in the chamber or the lysate or another solution can be used to resuspend the cells and pump them into a container, such as a reagent container containing the lysate, where the cells can be was lysed. In some embodiments, the lysate can be clarified by pumping the lysate through a filter membrane to remove unwanted cellular molecules and debris.

In some embodiments, pumping at least one process fluid into at least one bioprocessing chamber through at least one of the plurality of channels may further comprise extracting at least one biomolecule in a solid phase extraction membrane or washing the solid phase-bound material; and eluting the biomolecule from the solid phase. In some embodiments, pumping at least one process fluid into at least one bioprocessing chamber through at least one of the plurality of channels can further include precipitating and collecting biomolecules in the bioprocessing chamber housing the sedimentation filter.

In some embodiments, the bioprocessing method comprises: a) inserting at least one bioprocessing cartridge into a bioprocessing device, said bioprocessing cartridge comprising: i) at least one bioprocessing chamber containing a solid support therein; a plurality of mesoscale and/or microscale channels fluidly connected to the bioprocessing chamber; b) initiating a bioprocessing protocol on the bioprocessing device, the protocol comprising one or more of the following steps: i) controlling the pumps and valves on said bioprocessing cartridge, thereby providing reagents and/or samples from one or more containers to at least one bioprocessing chamber of each of at least one bioprocessing cartridge, ii) controlling said bioprocessing cartridge pumps and valves so that the reagents and/or samples are recirculated through at least one bioprocessing chamber of each of the at least one bioprocessing cartridge; and/or iii) controlling the pumps and valves on the bioprocessing cartridge valves to remove reagents and/or samples from at least one bioprocessing chamber of each of the at least one bioprocessing cartridge.

In some embodiments, the method of applying one or more fluids to a solid support may comprise: a) inserting at least one bioprocessing cartridge into a bioprocessing device, said bioprocessing cartridge comprising: i) at least one solid support contained therein and ii) a plurality of mesoscale and/or microscale channels in fluid communication with the bioprocessing chamber; and b) implementing a pumping sequence on the cartridge, wherein the pumping sequence Including entering one or more fluid addition cycles in which fluid is pumped from one or more containers in the bioprocessing device through a process fluid channel and into the chamber.

In some embodiments, the pumping sequence may also include entering a purge cycle after each fluid addition cycle, the purge cycle comprising pumping fluid from the bioprocessing chamber into a designated waste container. In some embodiments, the pumping sequence further includes entering a flow-through cycle after any fluid addition cycle, wherein the flow-through cycle includes opening a valve in a fluid flow channel connected to the bottom of the bioprocessing chamber and transferring fluid from the The bottom of the cavity is pumped through one or more fluid flow channels and into the top of the cavity. In some embodiments, the pumping sequence can also be initiated and terminated with a programmable controller.

In some embodiments, the pumping sequence performed includes entering one or more fluid addition cycles in which fluid is pumped into the cavity from a container in fluid communication with one of the process fluid connectors. The fluid in any container or the fluid added in any fluid addition cycle may be the same or different from the fluid in any other container or in any other fluid addition cycle. The pumping sequence may also include any fluid addition cycle followed by a purge cycle in which fluid in the chamber is pumped out of the chamber into a waste container or a container designated to collect waste fluid. In some embodiments, fluids from multiple reservoirs can be added during the same fluid addition cycle, or sequential fluid addition cycles can be performed without a purge cycle so that two or more fluids can be introduced into the chamber simultaneously ( Although the total volume of fluid added cannot exceed the volume available in the cavity). The pumping sequence may also include entering a flow-through cycle after any fluid addition cycle, wherein fluid in the chamber is pumped from the chamber through one or more process fluid channels of the cartridge and back into the chamber.

In some embodiments, the fluid addition cycle may be terminated after a predetermined amount of time has elapsed or after a predetermined volume of fluid has been added. Likewise, the purge and circulation cycles may be terminated after a predetermined lapse of time. The amount of time elapsed and/or the amount of fluid added depends on the protocol or type of biotreatment selected. In some embodiments, the protocol may include at least one flow-through cycle or incubation cycle, wherein the solid support is exposed to one or more process fluids in flow-through for a selected period of time, or in the fluid flow One or more process fluids to which one or more process fluids are exposed without circulation, for a holding period.

In some embodiments, the pumping sequence can be used for immunolabelling, washing, and incubation for western blot analysis. In such embodiments, a blotted membrane containing the separated protein, such as, for example, nitrocellulose or polyvinylidene fluoride (PVDF) membrane, is placed in the cassette chamber using a membrane holder, and the cassette is placed in In a bioprocessing device, the membrane holder provides fluid flow through the chamber and membrane without allowing the membrane to stick to any wall of the chamber. Antibody solutions can be added to the cartridge and membrane-containing chambers from the fluid containers of the bioprocessing device, along with appropriate blocking and washing solutions. The pumping sequence, duration and choice of solutions used depend on the specific analysis and proteins involved and can be modified by the user.

In some embodiments, the pumping sequence can be used to label molecules for western blot analysis with a label or tag, such as a fluorescent tag, such as a quantum dot, or a fluorescent dye, such as Alexa Fluor. dye. A solution containing a marker can be added to the cartridge from a fluid container of the bioprocessing device. The pumping sequence, duration and choice of solutions used depend on the specific analysis and proteins involved and can be modified by the user.

In another embodiment, the pumping sequence can be used for the preparation of transfection grade plasmids. Place the bacterial cells in solution or in cell culture medium into the sample container of the bioprocessing unit containing the appropriate bioprocessing cartridge. Cells are captured on the filter by pumping the device through the filter of the cartridge chamber. The cells are resuspended and lysed with a lysate such as one or more basic solutions, and the lysate is clarified by pumping it through another filter in a different cartridge chamber. Pass the clarified lysate through the solid phase extraction tray and into a waste container. The solid phase extraction disc is washed at least once before the bound biomolecules are eluted from the solid phase extraction disc. The eluted biomolecules are captured on a precipitation filter and washed at least once, at which point the precipitated biomolecules are dissolved in an appropriate buffer and pumped into the final product container of the bioprocessing unit.

In another embodiment, the pumping sequence can be used for food safety analysis. The sample can be placed in a sample container of a bioprocessing device containing an appropriate bioprocessing cartridge. By pumping the sample through the filter of the cartridge chamber with the device, the bacterial cells can be separated from larger debris and captured on the filter. Bacteria can be lysed with a lysate that is clarified by pumping it through another filter in a different cartridge chamber. The clarified lysate can be passed through the solid phase extraction tray and then into a waste container. The solid phase extraction disc is washed at least once before the bound DNA is eluted from the solid phase extraction disc and pumped into the final product container of the bioprocessing device.

In some embodiments, the method of applying one or more fluids to a solid support can also include independently opening and closing an inlet valve connected to a process fluid connector to selectively control flow into or out of a fluid flow channel. amount of fluid. In some embodiments, the method of applying one or more fluids to a solid support further comprises inserting a plurality of cartridges into a cartridge well, and performing a pumping sequence on each of said cartridges, wherein said The pumping sequence implemented on the cartridge is the same or different than the pumping sequence implemented on any other cartridge. In some embodiments, the pumping sequence performed on each cartridge is performed simultaneously.

In some embodiments, any of the bioprocessing methods described herein can be stored as part or all of a bioprocessing protocol on a bioprocessing device. In some embodiments, the stored protocol may also include details describing the timing of each step and the sequence of opening and closing valves and the pumping sequence of the pumps on the bioprocessing cartridges.

In some embodiments, the bioprocessing device and cartridge can be used as follows: the bioprocessing device is turned on and the various pressure and vacuum connections and supplies can be checked, one or more samples to be analyzed or bioassayed are inserted into a or multiple fluid container holders, if necessary, along with reagents in reagent containers and waste containers. One or more fluid container holders are placed in a removable fluid container tray and the tray is advanced into the bioprocessing device. One or more bioprocessing cartridges are inserted into the slots of the bioprocessing device and placed in fluid communication with the fluid containers in the fluid container holder. This fluid communication can be achieved through a fluid manifold in each tank or by inserting a suction/extraction tube connected to each bioprocessing cartridge into a fluid container. The slot holds the cartridge in the correct position and connects the control fluid manifold to the box. The device performs a system check to confirm that there are no security issues or connection errors. Using the GUI and input system, an operator selects a bioprocessing protocol from a catalog of stored protocols or can enter a protocol into an automated control system of the apparatus. The selected and entered protocol includes operating instructions for controlling the pumps and valves of the bioprocessing cartridge to implement one or more steps of the bioprocessing procedure. When the procedure is complete, the device may provide an alarm or signal notifying the operator that the procedure is complete. The cartridge can be removed from the device and discarded, and the fluid containers and fluid container holders removed with the removable fluid tray and cleaned and/or discarded if desired.

It will be appreciated that the above-described processes may be altered, including removing one or more steps, adding one or more steps, or changing the order of one or more steps, without departing from the scope of the methods described herein.

Referring to FIG. 1A , in some embodiments, a bioprocessing device 100 has a housing 10 constructed of any suitable material, such as plastic, metal, composite material, or any combination thereof, and is designed to accommodate Various components of the device. In some embodiments, housing 10 may comprise a sheet metal chassis covered with a plastic cover. The sheet metal enclosure can be any suitable sheet metal, such as an aluminum sheet base, which can provide structural integrity to the instrument, can limit EMI emissions, and can act as a heat sink for the power supply. The plastic cover can be constructed of any suitable plastic, such as urethane, and it can provide basic protection and an easy-to-wipe surface for internal components. The housing 10 comprises an opening 17 for access to the slot 13 in the device.

As shown in FIG. 1A , in some embodiments, the bioprocessing device 100 has two slots 13 into each of which a cartridge 12 has been inserted. Generally, the biological treatment unit may have any suitable number of tanks, such as about 1 to about 20 tanks, such as about 2 to about 15 tanks, about 3 to about 12 tanks, about 4 to about 10 tanks, about 6 to about 8 slots. In some embodiments, only one card 12 and one slot 13 are used during operation. In some embodiments, each slot 13 can be used with a cartridge 12 during operation. Typically, an operator can use the keyboard 18 to turn on the device and interact with the device, such as a computer control system or central processing unit included in the device, to select options and implement functions, or to provide and/or Or in response to connected instructions or queries, together with the graphical user interface (GUI) 15, the graphical user interface (GUI) 15 can provide the operator with status information, prompting information, scheme selection information, scheme occurrence information, scheme storage information and any other appropriate information. Additionally, the GUI can be used to override pre-run or running protocols if desired. GUI 15 may be any suitable display device, such as a liquid crystal display (LCD) touch screen, light emitting diode (LED) or electron ray tube (CRT).

Figure IB shows an alternative embodiment of a bioprocessing device 100 viewed from the front. The bioprocessing device 100 has a bioprocessing cartridge 102 inserted into a slot 112 . As shown in FIG. 1B , the bioprocessing device 100 also includes a GUI 104, a keyboard 106, a drawer 114 with a handle 108, each of which may be incorporated into or surrounded by the housing 110.

1C shows an alternative embodiment of a bioprocessing device 100 that includes a GUI 104 that displays information to a user, such as protocol information or status, run information, protocol details, protocol selections, and other options. In some embodiments, the device 100 can be equipped with 1, 2, 3, 4, or more than 4 tanks 112, which can be loaded and/or used simultaneously or concurrently. The bioprocessing device 100 may also include a keypad 106 on the GUI 104 for entering and selecting options on the device, and a drawer 114 with a handle 108, each of which may be incorporated into or surrounded by the housing 110 . FIG. 1D shows the bioprocessing device 100 of FIG. 1C with GUI 104 with keypad 106, drawer 114, handle 108, and surrounding base 110 viewed from the front.

Figure 2A shows a rear view of an embodiment of a bioprocessing device 200 with a cassette 210 inserted therein. The bioprocessing device 200 has a universal serial bus (USB) interface 220 that an operator or technician can use to download information from the device, for example, such as from a compatible storage device that can be installed on the device. Program operation information or device maintenance information of self-diagnostic software or firmware. Alternatively, USB interface 220 may be used to upload software or firmware updates and patches or other solutions to the computer control system in the device. Bioprocessing device 200 may also include an indoor or additional air connector 225 for communicating the device with an external air source, and a power connection 230 for communicating the device with a power source, such as a DC or AC power source. In some embodiments of the device 200, room air and/or room vacuum may be used instead of including a compressor and vacuum pump in the device.

FIG. 2B shows a rear perspective view of an alternative embodiment of the device with a cartridge 210 inserted into a slot 212 . The apparatus shown in FIG. 2B also shows a power switch 218, a power cord slot 235, and a utility connection 216 that can be used to provide air, vacuum, and/or other uses to the biological processing apparatus 200. FIG. 2C shows an optional rear view of biological treatment device 200 with USB port 216 , power cord slot 235 and optional reservoir drain connection 212 .

FIG. 3A shows a view of an embodiment of a keyboard 310 and a GUI 320 during a protocol selection step 330 of processing of an embodiment of a bioprocessing device. As shown, the user can pick to select one of several programs pre-loaded on the system. Any number of preloaded protocols can be included in the device and displayed on the GUI 320, including 1 or more protocols, 2 or more, 3 or more, 5 or more , 10 or more scenarios, or any other suitable number of scenarios that can be loaded into the memory of GUI 320. In some embodiments, a user may edit or modify preloaded protocols. Additionally, protocols with user-determined parameters can be entered into the GUI 320. The system may also include a time stamp 325. The keyboard 310 provides direction buttons 335 for navigating and navigating to different windows on the GUI 320 and selection buttons 340 for selecting options depending on the window and available commands/functions.

Figure 3B shows a view of a keypad 310 and GUI 320 embodiment of an embodiment of a bioprocessing device while a bioprocessing protocol run is in progress. As shown, the WesternBreeze protocol has been selected 335, the blocking step 337 has been completed, and the 30 minute primary antibody step 338 is in progress. With the runtime 343 setting of each further step, further steps 341 to be carried out can be displayed. Also functional on this screen are selection buttons 342 and 344 for pausing or stopping the process, respectively. As shown in FIG. 3B , other selection buttons 343 are provided which can be used to change step parameters, advance to the next step before the current step run time is complete, cancel a step, cancel a run, or can be used for any other suitable function. The duration of the entire protocol 350, the number of steps in the protocol 360 and or the remaining or elapsed time 355 for the current step can also be displayed on the screen of the GUI 320. It should be understood that the automated control system may provide for changing any of the following: step duration, step sequence, step type, number of steps, display options, alarm options, and input, run , control and record any other suitable parameters of any suitable scheme.

Figure 4 illustrates a partially disassembled view of an embodiment of a bioprocessing device 400 and associated components. The device 400 is shown having two slots 412 in which two cartridges 414 are ready to be positioned. Both cassettes may include at least 1, at least 2, at least 3, or at least 4 processing chambers 440 for each step of operation of the embodiments. Cassette 412 may also include a sipper 442 in communication, preferably fluid communication, with a fluid reservoir 444 of device 400 . Drawer 418 of device 400 may include handle 416, and drawer 418 is shown in an open position. Drawer slides 456 may be used to facilitate opening and closing of drawers 418 . In some embodiments, the slides 456 are located along the bottom of the drawer, or alternatively, may be located along the sides of the drawer. The drawer may have brackets 454 for proper alignment of the fluid reservoir tray 446 and waste container 448 with the suction portion 442 of the cassette 414 for proper alignment in the machine. The drawer in some embodiments houses at least one fluid reservoir tray 446 and a waste container 448 . Sample container 450 may be inserted into waste container 448 . A fluid reservoir tray 446 can also be placed in a waste container 448, and the fluid reservoir tray 446 can include at least one reagent reservoir 447 for containing and confining at least one reagent. The fluid reservoir tray 446 may further be configured to hold a collection tube 452 in which purified samples may be collected.

Figure 5 shows an embodiment of a bioprocessing device 500 with the outer housing removed. The bioprocessing apparatus 500 may include at least one vacuum reservoir 505 and/or at least one vacuum pump 510 for providing vacuum or suction as needed during processing to a bioprocessing cassette 515 having a vacuum chamber located in a tank 520. Suction/withdrawal tube 517 and cartridge holder 525 . Likewise, the bioprocessing apparatus 500 includes at least one pressure reservoir 530 and/or at least one air compressor 535 for providing air pressure to the bioprocessing cartridge 515 during processing. In some embodiments, at least one of vacuum reservoir 505, vacuum pump 510, pressure reservoir 550, and compressor 535 are in fluid communication with cassette 515 using tubing or other suitable vacuum or pressure connectors. In some embodiments, a diffuser can be connected to a vacuum or pressure connector to self-regulate the level of pressure/suction delivered to the cassette. The pressure or vacuum supplied to the cartridge can be controlled according to the configuration of the bioprocessing cartridge or can be a constant preset pressure (up to 50 psi) and vacuum (up to 20 Hg). In a specific example, the pumping mechanism of the bioprocessing unit has a pneumatic valve response time of 20 ms, an air reservoir capacity (vacuum and pressure) of 37.7 in ³ , an air compressor pump duty cycle (vacuum and pressure) of 50% duty ) and 80PSI air pump pressure.

As shown, the movable fluid container tray 540 can be movably engaged with a tray slide 545 that can help guide the tray 540 as it is moved into and out of the bioprocessing device 500 . A fluid container holder 550 can be received on the tray 540, which in the illustrated embodiment can include a waste reservoir 555, a container receptacle 560, and a container retention palate 562. As shown, the container 565 can be inserted into a suitably sized container slot 570 on the container holder 560, and a container securing plate 562 can be placed over some or all of the containers 565 to secure the container 565 in place, For example when pumping excess reagent or waste from fluid container holder 550 . The container base 560 also includes a waste reservoir access penetration 575 which, when aligned with the waste suction/extraction tube penetration 580 on the container mounting plate 562, provides a Suction/extraction tubes on one or more bioprocessing cassettes 515 enter waste storage tank 555 . In some embodiments, the fluid container holder 550 is configured such that the suction/extraction tubes located on one or more bioprocessing cartridges mate with appropriate containers or seats on the fluid container holder 550 to allow for the desired Biological treatment scheme. Optionally, in some embodiments, fluid container holder 550 can be configured such that the fluid manifold in bioprocessing device 500 can enter a suitable container or seat on fluid container holder 550 to allow for the desired Biological treatment scheme.

Although specific configurations are shown, it should be understood that fluid container holders may have any suitable configuration and that a variety of different types, sizes and And the configuration of the external container 565 and the container seat 560. For example, instead of providing a single integrated container holder for housing reagents and sample wells, multiple individual fluid container holders can be used and placed in removable fluid container trays, or can be used Custom sized fluid container holders for all fluid containers in a single tank. Likewise, fluid containers may or may not be provided to the fluid container holder, and when provided, one or more or all of the provided fluid containers may be empty, sterile, clean, prefilled and /or endotoxin-free. Additionally, in some embodiments, fluid container holders and some or all of the reagent and waste containers may be provided as part of a prepackaged kit if the user only needs to provide samples, if samples are provided in the protocol, or if required , providing one or more reagents.

Referring to FIG. 6 , a disassembled view of an embodiment of a fluid container holder 600 and a removable fluid container drawer 612 and fluid container tray 610 is shown. In some embodiments, the fluid container holder 600 may include a waste reservoir 615, The container seat 617 includes a container holder bottom 620 and a container holder top 625 and has a container slot 630 into which a container 635 can be inserted. The waste storage tank 615 includes a waste level sensor 616 that provides feedback to the automated control system in the bioprocessing plant, which can display a notification on the GUI and/or provide an audible alarm. The waste storage tank 615 may also include a fluid drain nozzle, which may be mounted on the housing of the biological treatment device through the penetration, and may be connected to a suitable drain or other waste fluid receptacle. The container holder 617 may include a waste reservoir inlet penetration 618 for fluidly communicating one or more waste lines from the bioprocessing cassette and/or from the fluid manifold with the waste reservoir 615 . The fluid container holder 600 may also include a container securing plate 640 . As shown, when combined with the appropriate containers and reagents and sample sets required for the desired bioprocessing protocol, the fluid container holder 600 can be placed in a removable fluid container tray 610, which can be moved like a drawer. Move into and out of the biological treatment unit.

Some embodiments Referring to FIG. 7, a fluid container holder 700 is provided that includes a container holder 710 that can also serve as a waste reservoir, a sample container 715, and a reagent reservoir tray 725. The sample container 715 can include a sample container cover 720, reagent storage The well tray 725 may include a plurality of reagent reservoirs 730 containing reagents as part of a prefilled reagent kit, or may include one or more empty reagent containers or empty reagent holders for holding reagents. In some embodiments, if all or some of the reagent reservoirs are pre-filled, the reagent trays may be fully or partially covered with aluminum, plastic, or any other suitable film before use or after inserting the trays into the device. The tray may be broken before or after it has been placed in the device. One or more of the reagents may be a buffer including, but not limited to, wash buffer, lysis buffer, neutralization buffer, resuspension buffer, precipitation buffer, or any other suitable buffer, or other suitable Reagents, such as antibodies, standards, blocking solution, deionized water. In some embodiments, the reagent can be ETOH, for example, 70% ETOH (70% ETOH with 30% NanoPure water), isopropanol, genome wash solution, genome extraction solution, ribonuclease, or any other Suitable reagents, rabbit antibodies, bovine serum albumin (BSA), standards such as MagicMark ^™ standards and chemiluminescent anti-antibodies such as Western Breeze Chemiluminescence anti-rabbit kit (Western Breeze Chemiluminescent kit-Anti-Rabbit). The system is adaptable for use with all chromogenic, chemiluminescent and fluorescent immunodetection reagents and protocols. In some embodiments, reagents can be provided as prefilled reagents in reagent vials and reagent bottles of suitable size. In some embodiments, the reagents may be added to reagent bottles and/or reagent vials provided in the kit included with the device. Fluid container cover 720 may include an inlet penetration to allow placement of a sample in sample container 715 and to allow processing of the sample on the bioprocessing cartridge. In some embodiments, the fluid container holder 700 is configured such that the suction/extraction tubes on one or more bioprocessing cartridges align with appropriate containers or seats on the fluid container holder 700 to allow for the desired biological treatment program. Optionally, in some embodiments, the fluid container holder 700 is configured so that the fluid manifold in the bioprocessing device can enter a suitable container or seat on the fluid container holder 700 to allow the desired bioprocessing to run. plan.

FIG. 8A shows an embodiment of a reagent reservoir tray 825 . The reagent reservoir tray 825 can have about 1 to about 15, about 1 to about 10, about 1 to about 7, about 1 to about 5, about 1 to about 3 reagent reservoirs 830 . In some embodiments, the reagent reservoir tray 825 can include at least 1 reagent reservoir, at least 2 reagent reservoirs, at least 5 reagent reservoirs, or at least 12 reagent reservoirs. The shape of the reagent reservoir 830 may be circular, square, polygonal, or any other suitable shape that accommodates a suitable volume of reagent to hold the desired amount of reagent. In some embodiments, the reagent reservoir tray 825 can also include at least one tube holder 835 into which a collection tube or container for collecting purified samples can be inserted. The tube holder 835 may be a hole into which a collection tube may be inserted or may be a structure that accommodates and supports a collection tube inserted therein. In some embodiments, a reagent reservoir tray may include more than one tube holder. In some embodiments, tube holder 835 can be configured to hold collection tubes with a volume of about 100 μL, about 1 mL, about 2 mL, about 5 mL, or about 10 mL tubes. The reagent reservoir 830 may also include at least one suction portion 840 located in the reagent reservoir, which is preferably in fluid communication with a portion of the bioprocessing cartridge. The suction portion 840 may be configured in any suitable configuration to allow reagents in the reagent reservoirs to collect on the suction portion 840 and thereby allow a substantial portion of the reagents to be introduced into the bioprocessing cartridge while reducing air bubbles entering the bioprocessing cartridge. In some embodiments, the reagent reservoir tray 825 can include an opening 831 to provide fluid communication with the waste tray.

FIG. 8B shows a perspective view of an alternative embodiment of a reagent reservoir tray 825 with an alternative embodiment/configuration of a reagent reservoir 830 . In some embodiments, the reagent reservoir tray 825 can include posts 827 for supporting and/or positioning the reagent reservoir tray 825 to the drawer and/or waste tray. As shown in Figure 8C, the reagent reservoir tray 825 can include at least one reagent reservoir 830 for containing and confining reagents, at least one tube holder 835, and at least one opening 831 to waste. In some embodiments, at least one reagent well 830 can also include a suction portion 840 configured to facilitate movement of reagents between individual reagent wells 830 of the bioprocessing cartridge and reagent well tray 825 . FIG. 8D is a side view of reagent reservoir tray 825 showing reagent reservoir 830 , tube holder 835 and suction portion 840 . Reagent reservoirs 830 may be substantially the same depth relative to one another or they may vary from one another. In some embodiments, the depth of individual reagent reservoirs 830 may depend on the length of the cartridge aspiration/withdrawal tubing. 8E is a cross-sectional view of reagent reservoir tray 825 showing tube holders 835 , reagent reservoirs 830 , and suction portions 840 at the bottom of each reservoir 830 . FIG. 8F is a close-up view of the suction portion 845 at the bottom of two different reagent containers 830 . Figure 8G illustrates an embodiment of the reagent well tray as viewed from the bottom of the tray, with a suction portion 840 protruding from the bottom of each reagent well 830, and an opening 831 leading to waste. The reagent reservoir tray can be configured to hold a suitable quantity and/or volume of reagents, and for example purposes only, the reagent reservoir tray 825 can include at least one collection tube slot or tube holder, TE buffer reservoir, and 70% Ethanol Reservoir, Isopropyl Reservoir, Elution Buffer Reservoir, Resuspension Buffer Reservoir, and Wash Buffer Reservoir.

9A and 9B show perspective views of opposite sides of an embodiment of a cartridge holder 900 with a bioprocessing cartridge 905 inserted therein. As shown, the cartridge holder 900 includes spring loaded housing bolts 910 that provide opposing support sides 915 through the interaction of support plates 925 and 930. Connection with 920. The support plate 930 may include individual supply connectors 932 (as shown in FIG. 9B ) as part of the manifold for providing control fluid to the bioprocessing cartridges. Individual supply connectors 932 may provide the connection of the control fluid manifold to the control fluid connectors on the bioprocessing cartridge 905 . The cartridge holder 900 may also include an inflatable bladder or bag 945, which may be connected to a pressure source via a bladder inflation/deflation line 950. Cartridge holder 900 may also include connection slots 955, which may provide connection of holder 900 in a slot of a bioprocessing device.

10A and 10B show a side view of a bioprocessing device cartridge holder 1000 . As shown, the cartridge holder 1000 includes a spring-loaded housing bolt 1010 that includes a spring 1012 for urging the support plate 1030 apart from the support plate 1025 while urging opposing retaining sides 1015 and 1020 closer together. Also shown in FIG. 10A is an inflatable bladder or bag 1045 in a deflated state. With bladder 1045 inflated, the resulting cartridge holder is shown in FIG. 10B , showing inflated bladder 1050 urging the two opposing retaining sides 1015 and 1020 as a whole against support plate 1030 , thereby compressing spring 1012 . In this manner, by holding the support plate 1025 stationary, the opposing holding sides 1015 and 1020 housings and bioprocessing cartridges held therein can be moved toward the support plate 1030 . In this way (and as shown in more detail in FIGS. 11A and 11B ), the control fluid connectors of the bioprocessing cartridges can be brought into fluid communication with the supply connectors on the support plate 1030, and the supply connectors can be connected to the control fluid manifolds. Tube. In some embodiments, pressure and vacuum can be provided to the bioprocessing cartridge to control the opening and closing of valves and control the activation of pumps to control the flow of fluid channels through the bioprocessing cartridge. It should be understood that other mechanisms for effectuating the connection between the control fluid connector and the supply connector may be used, including manual latches, locking latches, mechanically actuated connections, or electrical connections, among others.

11A and 11B show cross-sectional views of the embodiment of the cartridge holder 1000 shown in FIGS. 10A and 10B. As shown in FIGS. 11A and 11B , control fluid connector 1160 on bioprocessing cartridge 1105 does not engage supply connector 1132 on support plate 1130 when bladder 1145 is in the deflated state as shown in FIG. 11A . When bladder 1145 is inflated to form expanded bladder 1150, opposing retaining sides 1115 and 1120 and bioprocessing cartridge 1105 housed therein are moved toward support plate 1130 and supply connector 1132, thereby compressing gasket 1170 and A seal 1175 is formed between the supply connector 1132 , the gasket 1170 and the control fluid connector 1160 and places the supply connector 1132 in fluid communication with the control fluid connector 1160 .

FIG. 12 shows a disassembled view of a bioprocessing cartridge 1200 embodiment. Cassette 1200 has two film layers or foil layers 1202 and 1204 that may be sealed, such as heat sealed, outside of process fluid layer 1206 and outside of control layer 1208 of cartridge 1200 . The film layers 1202, 1204 may comprise any suitable plastic film, such as coated plastic film or suitable metal film, such as metal foil or coated metal foil, including aluminum foil, and the film may be coated with any suitable coating and and/or adhesive encapsulation, such as heat seal adhesives. In some embodiments, the film may be a PET film with a PE tie layer coated with a heat seal adhesive on the side that contacts the process fluid layer 1206 or the control layer 1208. In other embodiments, the film layer may comprise aluminum foil coated with a heat seal adhesive on the side that contacts the process fluid layer 1206 or the control layer 1208 . The membrane layers may include penetrations 1210 for control fluid connectors, penetrations 1212 for alignment guides 1214 for aligning process fluid layer 1206 and control layer 1208 . In some embodiments, the card can include a viewing access that provides a color indicator cavity. When sealed to the relevant surfaces of cartridge 1200 , film layers 1202 and 1204 may form a final barrier between process fluid channel 1220 on fluid layer 1206 and control fluid channel 1240 on control layer 1208 .

Cassette 1200 may include a process fluid layer 1206 having a plurality of process fluid channels 1220 and a plurality of penetrations 1222 through which control fluid connectors may be placed; and a plurality of penetrations 1224 for alignment guides. Process fluid channel 1220 may span the entire process fluid layer 1206 . Optionally, the process fluid channels are open on the front or outer surface of the layer and not open on the rear or inner surface of the layer. In this manner, process fluid channel 1220 may be isolated from control fluid layer 1208 and when applied to process fluid layer 1206 , membrane layer 1202 may effect shielding of process fluid channel 1220 in addition to providing layer channels or layer channel valves. Additionally, the process fluid layer 1206 may include an inlet valve compartment 1226, a process valve compartment 1227, a pump compartment 1228, a bioprocessing compartment 1230 with support ribs 1231, a color indicator compartment 1232, an inspection valve compartment Space 1233 , process fluid connector 1234 and gripper (gripper) 1236 .

Cassette 1200 may also include a control fluid layer 1208 having control fluid channels 1240 and control fluid connectors 1242 . Control fluid channel 1240 may span the entire control fluid layer 1208 or alternatively, may instead be open on the rear or outer surface of the layer and not open on the front or inner surface of the layer. In this manner, control fluid channel 1240 can be isolated from process fluid layer 1206 in addition to providing layer channels or layer channel valves, and in some embodiments, when applied to control fluid layer 1208, thin film layer 1204 can enable control fluid flow. Channel 1240 shielding. Additionally, the control fluid layer may include at least one of: inlet valve compartment 1246, process valve compartment 1247, pump compartment 1248, bioprocessing compartment 1250, color indicator compartment 1252, check valve compartment 1253, and clip device 1256.

Cassette 1200 may also include inlet valve membrane 1260 , process valve membrane 1261 , pump membrane 1262 and gasket 1264 between the inner surfaces of process fluid layer 1206 and control fluid layer 1208 . In some embodiments, cartridge 1200 can include at least one check valve membrane 1263 . In the illustrated embodiment, a suction/withdrawal tube 1266 is also included as part of the cassette 1200 . When assembled, inlet valve membrane 1260 is mounted in the compartment formed by inlet valve compartments 1226 and 1246 on process fluid layer 1206 and control fluid layer 1208 to form an inlet valve that is sealed by shrinking the membrane between the two layers. . Fluid can flow into the valve through process fluid channel 1220 connected to the process fluid side of the valve diaphragm, and the valve can be opened or closed with pressure or vacuum provided by control fluid channel 1240 on the fluid side of the valve diaphragm. In this way, by installing process valve membrane 1261 in the compartment formed by process valve compartments 1227 and 1247, a process valve can be formed, and by installing pump membrane 1262 in pump compartments 1228 and 1248, a pump can be formed. , by aligning bioprocessing compartments 1230 and 1250, a bioprocessing chamber can be formed, by aligning color indicator compartments 1232 and 1252, a color indicator chamber can be formed, if present, by installing check valve membrane 1263 in In the check valve compartments 1233 and 1253, check valves can be formed, and by placing a gasket on the control fluid connector 1242 and allowing a smaller opening in the control fluid connector penetration 1222 on the process fluid layer 1206, to To keep the gasket in place, the control fluid connector 1242 can be equipped with a gasket. As shown, in this embodiment, the bioprocessing chamber formed by bioprocessing compartments 1230 and 1250 may be open at the top for access by a user. Additionally, membranes 1202 and 1204 may be sealed such that the outer surfaces of process fluid layer 1206 and control layer 1208 seal fluid channels 1220 and 1240 and form a barrier to the channels.

When assembled, a bioprocessing cartridge may appear as shown in Figure 13, with Figures 13A and 13B showing front and rear views, respectively. As shown, the visible portion of cassette 1300 when assembled may include clamp 1305, bioprocessing chamber 1310 with support ribs 1312, membrane layers 1315 and 1320, color indicator chamber 1325, process fluid connector 1327, control Fluid connector 1330 , suction/withdrawal tube 1335 and slot alignment guide 1340 .

Additionally, a perspective view of the assembled bioprocessing cartridge of FIG. 13 is shown in FIG. 14 . As shown, the bioprocessing cartridge 1400 is viewed through the film layer 1405 on the surface of the process fluid layer, and the view extends through each preceding layer. Figure 14 shows process fluid channel 1410, inlet valve 1415, control fluid connector 1420, check valve 1430, color indicator chamber 1435, process valve 1440, pump 1445, alignment guide 1450, slot alignment on the process fluid layer Guide 1455, bioprocessing chamber 1460 with support ribs 1462, gripper 1465, control fluid channel 1470 on control fluid layer, and suction/extraction tube 1475.

15 shows a detailed view of an embodiment of a connection 1500 between an aspiration/extraction tube 1505 and a process fluid connector 1510 on a bioprocessing cartridge 1515. as the picture shows. The process fluid connector 1510 may be equipped with one or more retention rings 1520 which may provide corresponding retention grooves 1525 on the inner wall of the suction/extraction tube 1505 for a clearance fit. The process fluid connector 1510 includes one or more sealing grooves 1530 that provide an interference fit for a sealing ring 1535 on the inner wall of the suction/extraction tube 1505 . In this manner, retaining ring 1520 may secure suction/extraction tube 1505 to process fluid connector 1510 , while sealing ring 1535 may provide a seal for suction/extraction tube 1505 and process fluid connector 1510 . It will be appreciated that various alternative connections may be used to provide suction/extraction tubes connected to the cassette and that the tubes may also be integrally formed with the cassette.

FIG. 16A shows a disassembled view of a bioprocessing cartridge 1600 embodiment. Cassette 1600 has two film layers or foil layers 1602 and 1604 that may be sealed, such as heat or adhesive, to the outside of process fluid layer 1606 and the control layer or layer of cassette 1600. Inflatable layer 1608 outside. The film layers 1602, 1604 may comprise any suitable plastic film, such as coated plastic film or suitable metal film, such as metal foil or coated metal foil, including aluminum foil, and the film may be coated with any suitable coating and/or Adhesive encapsulation, such as heat seal adhesives. In some embodiments, the film may be a PET film with a PE tie layer wrapped with a heat seal adhesive in contact with the process fluid layer 1606 or with the inflation layer 1608 . In other embodiments, the film layer may comprise aluminum foil coated with a heat seal adhesive on the surface that contacts the process fluid layer 1606 or the gas-filled layer 1608 . The film layer may include penetrations 1603 for control fluid connectors, penetrations 1605 for alignment guides 1611, and in some embodiments, if present, penetrations to provide viewing access to the color indicator cavity department. Membrane layers 1602 and 1604 may form a final barrier for process fluid channels 1620 on fluid layer 1606 and inflation channels 1640 on control layer 1608 when sealed to the relevant surfaces of cartridge 1600 .

Cassette 1600 may include a process fluid layer 1606 having a plurality of process fluid channels 1620 . Process fluid channel 1620 may span the entire process fluid layer 1206, or alternatively, the process fluid channel may be open on the front or outer surface of the layer and not open on the rear or inner surface of the layer. In this manner, process fluid channel 1620 may be isolated from control fluid layer 1608 and when applied to process fluid layer 1606 , membrane layer 1602 may effect shielding of process fluid channel 1620 in addition to providing a layer channel or layer channel valve.

Cassette 1600 may also include an inflation layer 1608 having inflation channels 1640 and inflation connectors 1642 . The inflation channel 1640 may span the entirety of the inflation layer 1608 or alternatively, may instead be open at the rear or outer surface of the layer and not open at the front or inner surface of the layer. In this way, in addition to providing a layer channel or layer channel valve, the gas-filled channel 1640 can be isolated from the process fluid layer 1606 and when applied to the gas-filled layer 1608 , the membrane layer 1604 can achieve shielding of the gas-filled channel 1640 .

As shown, each gas-filled or control layer 1608 and process fluid layer 1606 includes bioprocessing chamber compartments 1603, 1605, 1607, and 1609, pump compartment 1610 and valve 1654, and inlet valve compartment 1618 and valve 1650, Process valve compartment 1644 and valve 1652, and in some embodiments, check valve compartment 1646 and valve 1656. In some embodiments, cassette 1600 can include a pass-through penetration 1673. Control layer 1608 may also include control fluid connectors 1642, which may include gaskets.

In some embodiments, a bioprocessing chamber can include filters 1662, 1664, 1666, and 1668, O-rings 1684, and gaskets 1685, 1687, as shown in the disassembled view of bioprocessing cartridge 1600. In some embodiments, the filter can be a Bla065 membrane, a nitrocellulose membrane, a glass fiber membrane, an Xthick membrane, a PPTR membrane, an anion exchange membrane, or any other suitable filter. In some embodiments, the filter of the bioprocessing chamber can be supported by a solid support or frit 1686,1688. The filter may be a single layer filter or a multilayer filter, and may be supported by one or more solid supports, for example as shown in bioprocessing chamber 1607 . The two layers of the cartridge and components found therebetween may be hermetically joined, such as by ultrasonic or other welding methods or with adhesives, latches, snap rings, or any other mechanism that joins the two plastic layers to form a bioprocessing cartridge implementation plan. In some embodiments, one or more bioprocessing compartments include one or more O-rings and/or tongue and groove assemblies to facilitate sealing. Additionally, in some embodiments, one or more bioprocessing chamber compartments on one or both of control layer 1608 and process fluid layer 1606 include structures, such as along one or both of their interior walls or within their A ridge on one or both of the inner walls to prevent or limit the interaction of the filter or solid support with the walls of the bioprocessing chamber. In some embodiments, rivets 1692 can be used to further support the membrane and seal the bioprocessing chamber.

FIG. 16B is a close up view of control fluid connector 1642 placed on inflatable layer 1608 . In some embodiments, the control fluid connector 1642 also includes a support 1693 that provides additional support for the control fluid connector during connection of the supply tube to the card. FIG. 16C is a side cross-sectional view of one half of bioprocessing chamber 1603 showing gasket 1685 , O-rings 1684 , 1686 and rivets 1692 , 1694 . Figure 16D shows a side view of the gas-filled layer 1608 and the fluid layer 1606 with the filter membrane 1683, O-ring 1684 and rivets 1692 creating additional support and/or holding the gas-filled layer 1608 and the fluid layer 1606 together.

17A and 17B show inside views of control layer 1701 and process fluid layer 1702 of a bioprocessing cartridge 1700 embodiment. As shown, each of control layer 1701 of FIG. 17A and process fluid layer 1702 of FIG. Inlet valve compartments 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738 and 1739, process valve compartments 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758 , 1760 and 1762 , check valve compartments 1764 , 1766 , 1768 , channel check valve compartment 1770 and channel penetrations 1772 , 1773 and 1774 . Control layer 1701 may also include control fluid connectors 1776 , control fluid channels 1794 and slot alignment guides 1796 . Process fluid layer 1702 may also include control fluid connector penetrations 1777 , process fluid connectors 1778 , 1779 , 1780 , 1781 , 1782 , 1783 , 1784 , 1785 , 1786 , 1787 , 1788 , and 1789 , and process fluid channels 1792 .

The control layer 1701 and/or the process fluid layer 1702 can have pump membranes, valve membranes, O-rings, and solid supports placed in associated compartments, and the two layers can then be hermetically joined, such as by ultrasonic or other welding methods or An embodiment of a bioprocessing cartridge is formed using adhesives, latches, clasps, clips, or any other mechanism that joins the two plastic layers. In some embodiments, one or more bioprocessing compartments include one or more O-rings and/or tongue and groove assemblies to aid in sealing. In some embodiments, each of bioprocessing chamber compartments 1703, 1704, and 1708 includes an O-ring. Additionally, in some embodiments, one or more bioprocessing chamber compartments located on one or both of control layer 1608 and process fluid layer 1606 include structures, such as along one or both of their interior walls or The ridges on one or both of their inner walls prevent or limit the interaction of the filter or solid support with the walls of the bioprocessing chamber.

Figures 18A and 18B show front and rear views 1801 and 1802, respectively, of an assembled bioprocessing cartridge embodiment. As shown, the bioprocessing cartridge includes bioprocessing chambers 1803, 1804, 1806, and 1808, pumps 1810, 1812, 1814, and 1816, inlet valves 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836 , 1838 and 1839, process valves 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860 and 1862, check valves 1864, 1866, 1868, channel check valves 1870, channels 1872, 1873 and 1874 , control fluid connector 1876, process fluid connector 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888 and 1889, control fluid seal 1890 and 1891, process fluid channel 1892, control fluid Channel 1894 and slot alignment guide 1896 .

Each of bioprocessing chambers 1803, 1804, 1806, and 1808 may include a solid support. In some embodiments, if the bioprocessing cartridge is used to purify and collect nucleic acids, such as DNA from whole cells, including plasmids or plasmid DNA, the bioprocessing chamber 1803 may include a cell separation chamber for separating whole cells from the cell culture medium. Filters, bioprocessing chamber 1804 may include a cell lysate clarification filter for clarifying the lysate by filtering out cell debris or other debris from the lysate, bioprocessing chamber 1806 may include solid phase extraction discs, cassettes or filters to reversibly bind nucleic acids from cell lysates, and bioprocessing chamber 1808 may include sedimentation filters to capture DNA eluted from solid phase extraction discs, cassettes, or filters. In some embodiments, one or more bioprocessing chambers include one or more O-rings and/or tongue and groove seals. In some embodiments, each of bioprocessing chambers 1803, 1804, and 1808 can include an O-ring or a gasket. Additionally, in some embodiments, one or more bioprocessing chambers include structures, such as ridges along or on one or both of their inner walls, that prevent or confine filters or solid supports Interaction with bioprocessing chamber walls.

As described elsewhere herein, each of the inlet valves 1818-1839, process valves 1840-1862, and check valves 1864-1868 may have pinched membranes in connected internal compartments relative to other bioprocessing cartridges , process fluid channel 1892 and control fluid channel 1894 may be fluid channels, such channels as described elsewhere herein, and process fluid connectors 1878-1889 may be process fluid connectors as described elsewhere herein. Control fluid connectors 1876 may be as described elsewhere herein, except that, in some embodiments, a single control fluid seal 1890 and/or 1891 is provided over multiple control fluid connectors and may be external rather than in Control fluid seals 1890 and 1891 are provided internally to the bioprocessing cartridge. The channel check valve 1870 can provide actively controlled fluid flow between the process fluid layer and the control fluid layer on the bioprocessing cartridge 1800 by allowing flow in only one direction between the layers. In some embodiments, the channel check valve 1870 can provide flow from the process fluid layer to the control fluid layer, while in other embodiments, the channel check valve 1870 can provide flow from the control fluid layer of the bioprocessing cartridge 1800 to the process fluid layer. flow. The channels can provide fluid communication in both directions between the process fluid layer and the control fluid layer on the bioprocessing cartridge 1800 .

The process and control fluid layers and the suction/extraction tubes of the bioprocessing cartridge can be made of any suitable material such as plastics such as ABS, polystyrene, polypropylene, polycarbonate and similar and can be injection molded Shaped or otherwise formed, such as by etching. In some embodiments, a bioprocessing cartridge can be injection molded and can have mesoscale fluid channels. In other embodiments, the bioprocessing cartridge may have microscale channels. The valve and pump membranes can be made of the same or different materials, and can be made of any material that is flexible enough to withstand the pressure and vacuum applied during bioprocessing. Examples of some suitable materials include thermoplastics and thermoplastic elastomers, such as SANTOPRENE ^™ and silicones. In addition, when the membrane is suitably thin to be flexible, the membrane can be made from a conventionally rigid material, albeit relatively generally rigid.

In some embodiments, one or more of the interior surfaces of a bioprocessing chamber may include bumps or may be frozen or otherwise surface modified to confine or prevent the solid support in the chamber from interacting with one or more Unwanted interactions of internal surfaces. In addition, other sealing surfaces or components may also be provided on or between the layers to assist in sealing individual parts of the box or the edges of the box when the box is assembled. For example, in some embodiments, bioprocessing cartridges may include one or more O-ring seals and/or tongues in grooves, or other sealing mechanisms may be included in layers around all or a portion of the cartridge edge, around In all or part of the layers of one or more biological processing chambers or in all or part of the layers surrounding one or more valves or pumps. Other sealing of the cassette layers may be achieved by sealing the layers together with an adhesive or by using ultrasonic or solvent welding or other suitable mechanism for sealing the layers together. Furthermore, although the bioprocessing cartridges described herein have been described as having two layers, it should be understood that the bioprocessing cartridges may have any suitable number of layers depending on the bioprocessing protocol used. For example, a bioprocessing cartridge may include multiple layers of process fluid, multiple layers of control fluid, or a temperature control layer through which the temperature of a portion of the cartridge is controlled or the temperature of all the cartridges is controlled. Also, in some embodiments, instead of providing pumps and valves with individual membrane layers extending across each of the individual compartments, providing individual membranes for the compartments as a single layer in the cartridge.

Figures 19A and 19B show an alternative embodiment of a bioprocessing cartridge. In some embodiments, the bioprocessing cartridge can include at least one bioprocessing chamber, which can include a filter cover for the bioprocessing chamber. For example purposes only, at least some of the bioprocessing chambers 1903 and 1904 may also include filter covers 1905, 1906, respectively, as shown in Figure 19A. In some embodiments, all bioprocessing chambers 1903, 1904, 1907, 1908 may include filter covers. In some embodiments, at least 1, or at least 2, at least 3, or more than 3 bioprocessing chambers include a filter cover. As shown in Figure 19A, a filter or membrane can be positioned on the fluid side 1901 of the bioprocessing cartridge and can be used to seal the bioprocessing chamber. In some embodiments, a filter can be positioned on the inflated side of the card, and a filter cap positioned over an individual bioprocessing chamber. As shown in Figure 19B, the inflatable side 1901 and fluid side 1902 can then be aligned and assembled together. The fluid side 1902 and inflation side 1901 are then assembled together by any suitable mechanism.

In some embodiments, at least one bioprocessing chamber can be modular as shown in Figures 20A and 20B. FIG. 20A shows a bioprocessing cartridge 2000 where two bioprocessing chambers 2003 and 2004 can be connected to the main body 2001 of the cartridge 2000 . In some embodiments of the bioprocessing cartridge, at least one bioprocessing chamber is modular and connectable to the bioprocessing cartridge. Figure 20A shows a bioprocessing cartridge 2000 with a modular bioprocessing chamber 2003 including a cell separation filter and a modular bioprocessing chamber 2004 including a cell lysate clarification filter. In some embodiments, the bioprocessing chambers 2003, 2004 may be located on opposite sides of the bioprocessing cartridge 2000, for example purposes, opposite the solid phase extraction tray 2006 as shown in FIG. 20A. In some embodiments, the modular bioprocessing chambers 2003, 2004 can be connected to the bioprocessing cartridge 2000 such that two modular bioprocessing chambers 2003, 2004 are connected to each other in series such that the first modular bioprocessing chamber is connected to the main body of the cassette, and then the second modular bioprocessing chamber is connected to the first modular bioprocessing chamber. In some embodiments, at least 1, at least 2, at least 3, or more than 3 cassette components can be modular. The modular assembly may include connectors 2011, 2013, 2015, and 2117, for example, male end connectors as shown in FIG. Figure 20B shows a close up view of a bioprocessing chamber 2003 attached to a bioprocessing card. FIG. 20B shows the bioprocessing chamber 2003 attached to the cartridge body 2001 . As shown in FIG. 20B , in some embodiments, the bioprocessing chamber 2003 can include male connectors 2011, 2013 of the modular bioprocessing chamber 2003 that mate with female connectors located in the bioprocessing cartridge body 2001. (female connector) 2019 and 2021 cooperate with each other. In some embodiments, the male connector is located in the body of the bioprocessing cartridge and the female connector is located on the bioprocessing chamber module.

Figures 21A and 21B show detailed views of a membrane holder, such as a blot membrane holder, that can be inserted into some embodiments of a bioprocessing cartridge. As shown in Figure 21A. Membrane holder 2100 may include a hinge 2110 connecting halves 2112 and 2114 of membrane holder 2100 that allows halves 2112 and 2114 to be opened or closed so that membrane 2116 may be inserted into the holder. The halves 2112 and 2114 may include ribs, such as diagonal ribs 2118, to support the retainer structure while allowing free flow of process fluid around the sides of the membrane 2116 and preventing or restricting the gap between the membrane 2116 and the walls of the bioprocessing chamber on the bioprocessing cartridge. mutual influence. FIG. 21B shows an alternative membrane holder 2120. In some embodiments, the holder may be folded along fold 2122 instead of a hinge, forming two parts 2121 and 2123 of the holder. Membrane holder 2120 includes fluid flow cutouts 2124 to provide uninterrupted access from the flow channels on the bioprocessing cartridge to the membranes in holder 2120 . Membrane holder 2120 may include support ribs 2128, such as oriented in a vertical orientation, to provide support to the holder while allowing free flow of process fluid around the sides of the membrane while restricting or preventing the bioprocessing chamber walls from contacting the holder. Interaction between membranes in vessel 2120. The membrane holder may be made from any suitable material, including plastics such as PVC, HDPE, polyester and APET, and may be injection molded or assembled from injection molded parts or may be die cut. In some embodiments, the membrane retainer helps provide process fluid laminar flow across both sides of the membrane in the bioprocessing chamber and prevents the membrane from sticking to or contacting one or more walls of the bioprocessing chamber.

Figure 22A shows an alternative embodiment of a blotted membrane holder 2200, which is similar to the blotted membrane holder 2120 shown in Figure 21B. Blot holder 2200 is folded in a similar manner to blot holder 2120, wherein holder 2200 is folded along crease 2222 to form two parts of holder 2200, part 2221 and part 2223, between which a blot membrane. Unlike holder 2120, holder 2200 includes, in addition to side fluid flow cutoffs 2224, bottom fluid flow cutoffs 2227 along folds 2222 to facilitate fluid flow around the imprinted membrane of holder 2200 and to facilitate Fluid flows between the bioprocessing cavity and the fluid flow channel on the bioprocessing cartridge. Similar to blotting membrane holder 2120, holder 2200 includes support ribs 2228, shown in a vertical orientation, to provide support for the holder while allowing free flow of process fluid around the sides of the membrane and restricting or Interaction between the walls of the bioprocessing chamber and the membrane in the holder 2200 is prevented. The blot holder 2200 can be made from the same or similar materials used to construct the other blot holders described herein.

Figure 22B shows an alternative embodiment of a blotted membrane holder 2250, which is similar to the blotted membrane holder 2200 shown in Figure 22A. Blot holder 2250 is folded in a similar manner to blot holder 2200, wherein holder 2250 is folded along crease 2252 to form two parts of holder 2250, first part 2251 and second part 2253, between which Blots can be placed between sections. Additionally, similar to holder 2200, holder 2250 includes, in addition to side fluid flow cutouts 2254, bottom fluid flow cutoffs 2257 along folds 2252 to facilitate fluid flow around and around the blotted membrane of holder 2250. The flow of fluid between the bioprocessing chamber and the fluid flow channel on the bioprocessing box is promoted. Unlike holder 2200, blot holder 2250 includes side fluid flow interrupters only on portion 2251 and not on portion 2253, and blot holder 2250 also includes bumps or nibs 2260, which facilitates the separation of the two parts 2251 and 2253 and provides a more consistent area for handling the blotted membrane. Similar to blotting membrane holder 2200, holder 2250 includes support ribs 2258, shown in a vertical orientation, to provide support for the holder while allowing free flow of process fluid around the sides of the membrane while restricting Or prevent the interaction between the walls of the bioprocessing chamber and the membrane in the holder 2250. The blot holder 2250 can be made from the same or similar materials used to construct the other blot holders described herein.

Figure 23 shows a basic flow diagram 2300 of one embodiment of a method for performing steps included in a bioprocessing protocol implemented with an automated control system on some embodiments of the bioprocessing apparatus provided herein. These steps are intended to be examples only, and some embodiments may include other steps, steps in a different order, and some of the steps shown may be omitted. As shown, the device can be primed 2310, the type and/or quantity of cartridges inserted into the cartridge slots of the device can be identified 2320, and a bioprocessing protocol can be selected 2330, which can be selected from a set of available preloaded protocols Or it may be generated on the device by the user by editing an existing protocol or by inputting a new protocol to the device or by uploading a protocol to the device. After protocol selection 2330, the device may prompt the user to confirm acceptance and correctness of the individual parameters associated with the protocol 2335, and the protocol may then be executed by the device on the desired cartridge 2340. In some embodiments, multiple protocols, multiple protocol types, and/or multiple cartridge types can be used on a device simultaneously or in a different order.

As an example, the embodiments of the bioprocessing cartridges disclosed with respect to Figures 12-14 can be used in conjunction with the bioprocessing devices described herein to perform any and all blocking, washing, Antibody Binding and/or Detection Steps. The following is an example of one embodiment of how such processing is performed. It should be understood that the following procedures are provided as examples only and that one or more steps may be modified and/or deleted, and that with the described bioprocessing apparatus and bioprocessing cartridges and other protocols and the same or different bioprocessing cartridge configurations, the For other types of biological treatment:

1) Prepare the bioprocessing device for processing by inserting the fluid container holder into the removable fluid holder tray and sliding it into the bioprocessing device. For each blotting membrane to be processed, the fluid container holder includes, a container containing the appropriate amount of blocking buffer, a container containing the appropriate amount of wash buffer, a container containing the appropriate amount of primary antibody, a container containing the appropriate amount of secondary antibody, and a container containing the appropriate amount of water . It should be understood that the fluid container holder provided to the user may be provided with one or more containers, which may be provided pre-filled or may be filled by the user. In some embodiments, at least one container is prefilled, while in other embodiments, all or none of the containers are prefilled.

2) Insert the blotted membrane to which the protein to be detected has been transferred into the blotted membrane holder and enter the bioprocessing chamber of the bioprocessing cartridge configured as described in FIGS. 12-14 through the open top of the bioprocessing chamber. Multiple cassettes can be used, each with its own blotting membrane, blotting membrane holder, and fluid container in the fluid container holder.

3) Insert the bioprocessing cartridge into the groove of the bioprocessing device and fix it to the cartridge holder. The fluid manifold of each cartridge is connected to the control fluid connector of the cartridge by inflating the bladder associated with each cartridge holder. The operator ensures that the suction/withdrawal tube is placed in the proper container on the fluid container holder.

4) The bioprocessing device can confirm that the trays and cassettes are inserted correctly and that the cassettes have not been used before.

5) The operator selects the desired protocol for the cassette on the automated control system and activates it. Ensure that all inlet valves are closed at this time by verifying their activation status.

The remainder of this example procedure will be described for a single box, and once a scheme is selected, will be automatic and hands-off:

6) An automated control system, using pressure and/or vacuum through a suitable control fluid channel, activates the membrane of the inlet valve associated with the closed buffer container to open and activate the lower central portion connected to the bioprocessing chamber on the cassette A pump and process fluid valve associated with the flow channel of the ("central valve") to pump the blocking buffer out of the blocking buffer container and into the bioprocessing chamber. After the blocking buffer has been pumped into the bioprocessing chamber, the blocking buffer inlet valve is forced to the closed position.

7) withdrawing the blocking buffer through one of the process fluid valves associated with the flow channel into the side of the bioprocessing chamber ("side valve") by activating the pump, then pumping the withdrawn fluid back into the bioprocessing chamber through the center valve, and Recirculate the blocking buffer through the bioprocessing chamber and the blotting membrane. Either side valve can be used, depending on the size of the blot. Said recycling can occur as follows. Open the side valve and pump buffer through the side valve and associated flow channel to the pump. Close the side valves and open the center valve. The pump is started, blocking buffer is pumped from the pump through the center valve to the bioprocessing chamber, and the center valve is closed and the procedure repeated for the time selected by the protocol. Each return of fluid to the bioprocessing chamber causes slight movement of the blotting membrane and may cause localized swirl formation or vortex formation, which ensures sufficient or substantially uniform exposure of the surface of the blotting membrane to blocking buffer.

8) Once closure is complete, pump the buffer from the chamber by selectively opening the center valve, pumping fluid to the pump, closing the center valve, opening the waste container inlet valve, and pumping fluid to the waste container Pass through center valve and pump into waste container on fluid container holder.

9) In this way, the blot can be washed, the primary antibody can be incubated with the membrane, the membrane can be washed again, the secondary antibody can be incubated with the membrane, the membrane can be further washed and rinsed, all following an automated protocol, without User cooperation is required. Any or all of the above steps may include recirculation of the process fluid as described above. After the final rinse, the device can provide an alert that it is complete, and the cartridge can be removed from the device, and the blot removed from the cartridge for further processing, such as protein detection and analysis.

Antibodies, blocking buffers, washing buffers, and developing/detecting solutions may comprise any suitable components for use in immunoblotting procedures without limitation. Antibodies, blocking buffers, and wash buffers may be provided commercially individually, as components of kits, or may be prepared by the end user. As a non-limiting example, antibodies used in accordance with the presently described embodiments may include one or more primary antibodies, one or more secondary antibodies, or a combination of one or more primary antibodies and one or more secondary antibodies. combination.

Suitable primary antibodies may include any antibody selected by the user for use in the presently described system. In some embodiments, the primary antibody can be raised against a user-defined antigen. In some embodiments, the primary antibody may be a complex mixture of antibodies recognizing multiple antigens. Primary antibodies are commercially available or can be prepared by the user.

Primary antibodies can be polyclonal or monoclonal. Monoclonal antibodies can be raised in mice or rats. Monoclonal antibodies can be of IgG (IgG1, IgG2a, IgG2b, IgG3), IgM, IgA, IgD and IgE subclasses. Polyclonal antibodies can be raised in rabbits, mice, rats, hamsters, sheep, goats, horses, donkeys or chickens. In embodiments, the antibodies may be from human serum. Human antibodies can be at least partially purified or fully purified. Methods of making and purifying antibodies are well known in the art. General guidance on the preparation, purification and use of various antibody preparations can be found, for example, in the reference textbook Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Sping Harbor, New York, Harlow et al. , 1999, Using Antibodies: A Laboratory Manual (using antibodies: a laboratory manual), Cold Sping Harbor Laboratory Press, NY, and Harlow, et al., 1988, In: Antibodies, A Laboratory Manual (antibodies: a laboratory manual), Cold Sping Harbor, NY , all of which are hereby expressly incorporated by reference in their entirety.

In some embodiments, the primary antibody may be a "loading control antibody." The loading control antibody can be provided by the user, or can be provided commercially as part of the presently described system. Exemplary but non-limiting loading control antibodies that can be used or provided with the presently described systems and methods can include anti-actin, tubulin, histone, vimentin, lamin, GAPDH, VDACl, COXIV , hsp-70, hsp-90 or TBP antibodies.

The concentration of the primary antibody in the immunoblotting buffer will undoubtedly vary, depending on the specific primary antibody used, the context in which the antibody is used, and various other properties inherent to the antibody. Determining the appropriate primary antibody concentration to use in any given experimental protocol is within the skill of the art. Typically, the primary antibody concentration is 1:10-1:20,000, 1:100-1:15,000, 1:1,000-1:10,000 or 1:1,500-1:5,000.

Suitable secondary antibodies for use in the presently described systems and methods include any antibody that recognizes and binds to the primary antibody. Optionally, secondary antibodies can be conjugated to one or more means of detection. It will be apparent to those skilled in the art that the composition of a suitable secondary antibody may of course vary depending on the nature of the primary antibody or antibodies with which it is used. Typically, the secondary antibody chosen binds at least a portion of the primary antibody used. The choice of an appropriate secondary antibody also depends on the method used to detect the signal in subsequent steps. If the end user detects the analyte using chemiluminescent techniques, a suitable secondary antibody can be conjugated, for example, to peroxidase. If the researcher is using colorimetric techniques to detect the analyte, a suitable secondary antibody can be conjugated to, for example, alkaline phosphatase. If researchers are using fluorometric techniques to detect analytes, suitable secondary antibodies can be conjugated to fluorophores (including but not limited to FITC, TRITC, Texas-Red, Alexa-Fluor reagent, quantum dots, semiconductor nanocrystals, etc.) ) coupling. Optionally, a secondary antibody can be conjugated to one or more biotin moieties, and a detection molecule (eg, peroxidase, phosphate, fluorophore, etc.) can be conjugated to avidin or streptavidin. With this biotin/avidin system, weak signals can be amplified in some cases. Typically, the concentration of the secondary antibody is 1:10-1:20,000, 1:100-1:15,000, 1:1,000-1:10,000 or 1:1,500-1:5,000.

Suitable secondary antibodies for use in the presently described systems and methods can be raised, for example, in rabbits, mice, rats, hamsters, pigs, sheep, goats, horses, donkeys, turkeys or chickens. Secondary antibodies are usually raised in a different species than the primary antibody was raised in. A secondary antibody can be generated such that it recognizes and binds to a portion of the primary antibody. Secondary antibodies can be at least partially affinity purified. The secondary antibody can be anti-mouse IgG, mouse IgA, mouse IgM, rat IgG, rat IgA, rat IgM, rabbit IgG, rabbit IgA, rabbit IgM, hamster IgG, hamster IgA, hamster IgM, goat IgG , goat IgA, goat IgM, horse IgG, horse IgA, horse IgM, sheep IgG, sheep IgA, sheep IgM, donkey IgG, donkey IgA, donkey IgM, chicken IgG, chicken IgA, chicken IgM, chicken IgY, human IgG, human IgA or human IgM. The secondary antibody may be coupled to one or more detection molecules, such as alkaline phosphatase, peroxidase, biotin, fluorophores or quantum dots or semiconductor nanocrystals, as examples.

A blocking buffer may include a suitable blocking reagent dissolved or dispersed in a diluent. As non-limiting examples, suitable blocking reagents may include whole serum, fractionated serum, bovine serum albumin, casein, soy protein, non-fat milk, gelatin, fish serum, goat immunoglobulin, rabbit immunoglobulin, mouse Immunoglobulin, rat immunoglobulin, equine immunoglobulin, human immunoglobulin, porcine immunoglobulin, chicken immunoglobulin, or synthetic blocking reagents such as those available from, for example, BioFX Laboratories, Kem-En-Tec Diagnostics or GeneWay Blocking reagents such as those commercially available from Biotech. A variety of commercially pre-prepared blocking reagents are available, all of which can be provided in the kits described herein. Such commercially available blocking reagents include, but are not limited to, for example, WesternBreeze, I-BLOCK, BLOCKIt, PerfectBlock, Synthetic Blocking Buffer (synthetic blocking buffer, BioFX Labs), GelantisBetterBlock, SeaBlock, StartingBlock, and Protein-Free Blocking Buffer ( Protein-free blocking buffer, Pierce). In provided embodiments where the blocking reagent is dissolved or dispersed in the diluent, the blocking reagent is present in an amount ranging from about 0.1 wt.% to about 50 wt.%, about 1 wt.% to about 40 wt.%, about 2.5 wt.% to about 25 wt.%, about 5 wt.% to about 15 wt.%, or about 10 wt%. In embodiments, the amount of blocking reagent present in the immunoblotting buffer can be up to about 75 mg/ml, up to about 50 mg/ml, up to about 40 mg/ml, up to about 30 mg/ml, up to about 20 mg/ml, up to about 15 mg /ml, up to about 10 mg/ml, up to about 5 mg/ml, up to about 2.5 mg/ml, up to about 1 mg/ml, up to about 0.5 mg/ml, up to about 0.25 mg/ml, or up to about 0.1 mg/ml. Suitable diluents that may be used as carrier media for blocking reagents include any aqueous buffer having substantially physiological pH and ionic strength. Exemplary, but non-limiting, diluents may include phosphate ions, bicarbonate, TAPS, Bicine, Tris, Bis-Tris, Tricine, HEPES, TES, MOPS, PIPES, Cacodylate, Any buffer of MES, acetate, ADA, ACES, ethanolamine, BES, acetamidoglycine or glycinamide. Exemplary buffers suitable for use as diluents may include, but are not limited to, eg, PBS, Hank's solution, TBS, TE, TEN, and the like. Optionally, the diluent may include a detergent. Suitable detergents may include non-ionic, non-denaturing detergents such as Triton X-100, Triton X-114, NP-40, Brij-35, Brij 58, Tween-20, Tween-80, Octyl Glucosides and octylthioglucosides and detergents such as thiobetaines, including SB-12, SB-14 and SB-16. The diluent may contain from about 0.01% by volume to about 5% by volume, from about 0.05% by volume to about 2% by volume, from about 0.1% by volume to about 1.5% by volume, or from about 0.5% by volume to about 1% by volume of detergent.

In some embodiments, a suitable wash buffer may be the same diluent used to disperse the blocking reagent as described above. In some embodiments, a wash buffer may be a diluent lacking one or more of its components. In some embodiments, the wash buffer may be a diluent lacking a blocking reagent.

In some embodiments, a wash buffer or diluent may be provided in full strength (ie, 1X strength) or may be provided as a concentrated solution to facilitate its storage or transportation. The concentrated wash buffer or diluent can be diluted by the user with, for example, deionized water, sterile water, or any other suitable diluent. Concentrated wash buffers/diluents are provided at up to about 50X, up to about 25X, up to about 20X, up to about 10X, up to about 5X, or up to about 2X strength.

In some embodiments, the wash buffer/diluent provided to the user may be in one or more plastic or glass bottles provided with the kit. Each kit can include 1-10 vials of wash buffer, 1-5 vials of wash buffer, or 1-2 vials of wash buffer. Each bottle of diluent may contain up to 5L, up to 4L, up to 3L, up to 2L, up to 1L, up to 500ml or up to 100ml of diluent.

As an example, the bioprocessing cartridge embodiments described in FIGS. 16-18 can be used in conjunction with the bioprocessing devices described herein to perform cell separation, lysis, clarification, binding, washing, elution, precipitation, and/or nucleic acid processing. Or a collection step, the nucleic acid processing includes nucleic acid collection processing such as DNA or fragments thereof including plasmid DNA, genomic DNA, viral DNA and bacterial DNA or fragments of any of the above. An example of one embodiment of how such processing is carried out follows. It should be understood that the following procedures are provided as examples only and that one or more steps may be changed and/or deleted, and that other types of biological treatment:

1) Prepare the bioprocessing device for processing by inserting a fluid reservoir holder for each sample to be processed into the removable fluid holder tray and sliding into the bioprocessing device. Each fluid reservoir holder includes a sample container into which a sample is inserted, a waste container, an RNase (ribonuclease) reservoir, a resuspension buffer reservoir, a lysis buffer reservoir, a neutralization buffer reservoir, a wash Reservoir, Eluent Reservoir, Isopropanol Reservoir, Ethanol Reservoir, Collection Buffer Reservoir, and Collection Reservoir, each containing the appropriate amount of the relevant solution (or empty, for e.g. waste container and collection container). The fluid reservoir holder is provided with either an empty reservoir or a prefilled reservoir with a sample to be added. In some embodiments, one or more reservoirs can be pre-filled, while one or more reservoirs can be filled by the user. In some embodiments, fluid reservoir holders are provided with at least one pre-filled reservoir, while in other embodiments, fluid reservoir holders are provided without a reservoir and/or provided with one or more The storage pool is empty.

2) For each sample to be processed, a bioprocessing cartridge is inserted into the slot of the bioprocessing device and secured to the cartridge holder. The fluid manifold of each cartridge is connected to the control fluid connector of the cartridge by inflating the bladder associated with each cartridge holder. When inserting the cartridge into the slot and cartridge holder, the operator ensures that the suction/withdrawal tubes are placed in the proper receptacle on the fluid container holder.

3) Bioprocessing unit can confirm correct insertion of trays and cassettes.

4) The operator selects the desired protocol for the cassette, which may be pre-programmed in the host unit or may be user-definable on the automated control system, and activates it. At this point ensure that all inlet valves are closed by verifying their activation status.

5) Referring to the reference numbers in Figures 18A-B, the remainder of this example procedure will be described for a single cartridge, and once a protocol is selected, it will be automatic and hands-off:

6) The automated control system opens the membrane inlet valve 1818 associated with the sample with pressure and/or vacuum through a suitable control fluid channel, activates the pump 1810, and pumps the sample through the cell separation bioprocessing chamber 1803 connected to the cartridge Process fluid valves 1846 and 1852 associated with the process fluid channel at the inlet at the upper central portion, pumped through the filter membrane in chamber 1803, through the outlet of chamber 1803 at the bottom central portion of the chamber located behind the cassette or on the control fluid layer The cavity, through channel 1872, and back into the process fluid layer of the cartridge, exits through inlet valve 1820 and is pumped into a waste container on the fluid container holder, actuating the appropriate valves to open and close as needed to allow fluid to pass through the appropriate flow in the correct process fluid channel and prevent fluid from flowing into the wrong process fluid channel at the wrong time. The cells of the sample are captured on a filter in chamber 1802 .

7) Remove media remaining in cavity 1803 and process fluid passages leading cavity 1803 and pump 1810 by blowing air, such as about 30-40 psi pulses of air or continuous flow, from the control fluid layer, through Check valve 1868 to process fluid layer, through pump 1810, process fluid valves 1846 and 1852, through filter in bioprocessing chamber 1803, through chamber 1803 at bottom center portion of chamber 1803 located behind cassette or on control fluid layer The outlet of C1 exits cavity 1803, passes through channel 1872, and returns to the process fluid layer of the cartridge, and is pumped out through inlet valve 1820 and into a waste container on the fluid container holder.

8) Using pump 1810, pump RNase from RNase reservoir through inlet valve 1822 into pump 1810, through inlet valve 1824 and into resuspension buffer reservoir.

9) Use the pump 1810 to resuspend the cells on the filter membrane in the chamber 1803 by pumping the resuspension buffer (with RNase) through the inlet valve 1824, through the pump 1810, through the process valve 1840 and the channel 1872, where the resuspension The buffer is moved to the control fluid layer, pumped into the chamber 1803 through an outlet at the bottom center portion of the chamber 1803 located either behind the cartridge or on the control fluid layer (this time serving as an inlet), and through the filter (to match the step 6-7, the opposite direction of cell separation), pump out the chamber 1803 through the inlet of the central part of the cell separation biological treatment chamber 1803 (used as an outlet at this time), and pump into the lysis buffer storage tank through the process valve 1852 and the inlet valve 1826 .

10) Remove any cells remaining in chamber 1803 and the process fluid pathway leading from chamber 1803 to the lysis reservoir by blowing air, such as about 30-40 psi pulses of air or a continuous stream of air, from the Control fluid layer, through check valve 1868 to process fluid layer, through process valve 1840 and channel 1872, where it is moved to control fluid layer, through bottom center portion of chamber 1803 located behind or on control fluid layer pumped into the chamber 1803 and passed through the filter (in the opposite direction to the cell separation in steps 6-7), through the inlet at the upper central part of the cell separation bioprocessing chamber 1803 (used at this time) as an outlet) pumped out of chamber 1803, through process valve 1852 and inlet valve 1826 into the lysis buffer reservoir.

11) Lyse the cells by gently mixing the solution in the lysis buffer reservoir back and forth between the lysis buffer reservoir and the resuspension reservoir by pumping the cells from the lysis buffer reservoir with pump 1810 to Through inlet valve 1826, through process valve 1846, through pump 1810, through inlet valve 1824, and into resuspension buffer reservoir and back. This back and forth can occur as needed to lyse the cells, and the solution can end up in either reservoir.

12) Assuming the lysed cells are in the resuspension buffer reservoir, pump the neutralization buffer from the neutralization buffer reservoir with pump 1810 through inlet valve 1828, through process valve 1846, through pump 1810, through inlet valve 1824, And pump into the resuspension buffer storage tank. The solution can be mixed by pumping back and forth through the route and, if the layers formed are allowed to separate, can end up in either reservoir.

13) Assuming the lysed and neutralized cells are in the resuspension buffer, the lysate can be clarified by pumping the lysate through the inlet valve 1824 with pump 1810, through the pump 1810, through the process valve 1848, through the top center of the chamber Inlet at , pumped into bioprocessing chamber 1804, through clarification filter in chamber 1804, where cell debris and other debris are removed, pumped out of chamber 1804 through an outlet at the bottom central portion of the chamber on the control fluid layer, Through channel check valve 1870 to the process fluid layer, through check valve 1866 (which valve prevents access to the control fluid layer), through channel 1874 back to the control fluid layer, pumped into the bioprocessing chamber 1806 through the bottom center inlet, through the DNA binding The solid phase extraction filter, membrane, disc or cartridge is pumped out of the bioprocessing chamber 1806 through an outlet located at the top central portion of the chamber 1806 above the process fluid layer, through the process valve 1858 and the inlet valve 1818, into the sample container, which is currently as a waste container.

14) Wash the solid phase extraction filter, membrane, disc or cartridge by pumping wash solution from the wash solution container with pump 1812 through inlet valve 1830, through pump 1812, through process valve 1850, through check valve 1870 and 1866, check valves 1870 and 1866 prevent wash fluid from passing through them, through channel 1874 to the control fluid layer, pumped into bioprocessing chamber 1806 through the bottom central inlet, through DNA binding solid phase extraction filters, membranes, discs or cassettes, through Outlet at the top center portion of chamber 1806 above the process fluid layer, pumps out of bioprocessing chamber 1806 through process valve 1858 and inlet valve 1818 into a sample (waste) container.

15) After the washing step, any residual washing fluid is removed from the bioprocessing chamber 1806 by blowing pulses of air, such as about 30-40 psi or a continuous stream of air, from the control fluid layer, through check valve 1866 to the process fluid layer, through penetration 1874 where air is diverted to the control fluid layer, passes through the filter through an inlet at the bottom central portion of chamber 1806, and is pumped out of bioprocessing chamber 1806 through an outlet at the upper central portion of bioprocessing chamber 1806 , through process valve 1858, and through inlet valve 1818 into the sample (waste) container.

16) Elute the bound DNA from the solid phase extraction filter, membrane, disc or cassette by pumping the eluate solution from the eluate solution reservoir with pump 1812, through inlet valve 1832, through pump 1812, through Inlet valve 1850, through check valves 1870 and 1866, check valves 1870 and 1866 prevent eluent from passing through them, through channel 1874 to control fluid layer, pumped into bioprocessing chamber 1806 through bottom central inlet, through DNA binding solid phase extraction Filters, membranes, discs or cartridges, pumped out of bioprocessing chamber 1806 through an outlet located at the top central portion of chamber 1806 above the process fluid layer, through inlet valve 1860, through pump 1814, through inlet valve 1834 into isopropyl Alcohol storage pool.

17) Mix the solution in the isopropanol container by gently mixing the solution in the isopropanol container back and forth between the isopropanol container and the eluent solution container, using pumps 1814 and 1812 to move the solution from the isopropanol The propanol container is pumped through inlet valve 1834, through pump 1814, through process valve 1854, through pump 1812, through inlet valve 1832 and into the eluent container, and back again. This back and forth can occur as needed to mix the solution, and the solution should end up in the isopropanol container.

18) Capture DNA on a precipitator in bioprocessing chamber 1808 as follows: With pump 1814, pump the solution in the isopropanol container through inlet valve 1834, through pump 1814, through inlet valve 1856 And through the inlet of the upper part of the biological treatment chamber 1808, pumped into the chamber 1808, passed through the settler filter in the chamber 1808, pumped out from the bottom outlet of the chamber 1808 on the control fluid layer, passed through the channel 1873, passed through the process valve 1863 and Inlet valve 1818 and pump into sample (waste) container.

19) Wash the precipitator with ethanol by pumping the ethanol from the ethanol container with the pump 1814 through the inlet valve 1836, through the pump 1814, through the inlet valve 1856 and through the upper inlet of the bioprocessing chamber 1808, into the chamber 1808, Pumped from the bottom outlet of chamber 1808 above the control fluid layer through the settler filter in chamber 1808, through channel 1873, through process valve 1863 and inlet valve 1818 and into the sample (waste) container.

20) Drying of the precipitator by blowing air such as about 30-40 psi air pulses or continuous flow from the control fluid layer, through the check valve 1864, to the process fluid layer, through the process valve 1862, through the biological process Inlet on upper part of chamber 1808, pumped into chamber 1808, through settler filter in chamber 1808, pumped out from bottom outlet of chamber 1808 at control fluid layer spread, through passage 1873, through process valve 1863 and inlet valve 1818 Pump into sample (waste) container.

21) Elute the DNA from the pellet into the collection container by pumping the collection buffer from the collection buffer container with pump 1816, through inlet valve 1838, through pump 1816, through check valve 1864, check Valve 1864 prevents collection buffer from passing through it, through process valve 1862, through an inlet in the upper part of bioprocessing chamber 1808, pumped into chamber 1808, through a settler filter in chamber 1808, and out the bottom of chamber 1808 at the control fluid layer Pumped out, through channel 1873, through inlet valve 1838 into a collection container.

It should be understood that this program is for example only and should not be considered limiting in any way. A variety of different procedures can be performed using the cassette of Figures 18A-B, and the cassette can be configured in different ways, according to different protocols, including with additional or fewer bioprocessing chambers, different spatial orientations of any bioprocessing chambers , inlets and outlets of bioprocessing chambers, different flow channel configurations, different numbers, types and spatial orientations of pumps and valves, different numbers and types of containers and process solutions, and different types of process samples.

Depending on the protocol implemented, any suitable buffer, wash, resuspension buffer, lysis buffer, neutralization buffer, RNase, elution/collection buffer or precipitation buffer may be used with or without any other suitable reagent. Suitable buffers, as well as their components and methods of use, are described in the following U.S. patent references: 6,914,137, 2006/0154247, 2007/0117972, 6,242,220, 5,990,301, 7,214,508, 7,109,322, and 6,297,371, all of which, as indicated herein, incorporated by reference.

According to the presently described systems and methods, any suitable biologically acceptable buffer having a pH of about 3.5 to about 9, about 5 to about 8, about 6.5 to about 7.5 is suitable for use in preparing the resuspension buffer, for example, Tris, TAPS, Bicine, Tricine, HEPES, TES, MOPS, PIPES, Formazinate, MES, Acetate, etc. In some embodiments, the resuspension buffer may comprise 1 mM-100 mM, 5 mM-50 mM, or 10 mM-20 mM chelating agents such as EDTA, EGTA, ALA, BAPTA, defarasirox, deferiprone, deferox Amines, DTPA, dimercaptopropanol, DMPS, DMSA, etc. In some embodiments, the resuspension buffer may optionally comprise RNases such as endoribonucleases and exoribonucleases, including RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM , RNase T1, RNase T2, RNase U2, RNase V1, RNase V, PNPase, RNase PH, RNase II, RNase R, RNase D, RNase T, exoribonuclease I, exoribonuclease II, etc. In some embodiments, the RNase A formulation may comprise 2.4 mg/ml RNase A, 50 mM Tris-HCL, pH 8.0, and 10 mM EDTA. In some embodiments, the resuspension buffer may optionally comprise lysozyme. In some embodiments, the resuspension buffer may optionally comprise about 1 mM to about 500 mM, about 10 mM to about 200 mM, about 20 mM to about 100 mM, about 30 mM to about 75 mM carbohydrates, such as sugars. Exemplary sugars include glucose, fructose, galactose, mannose, maltose, lactose, and the like. An exemplary resuspension buffer may include an aqueous solution containing 50 mM Tris, pH 7.4, 100 μg/ml RNase Al, 10 mM EDTA, and 5 mM glucose. In some embodiments, the resuspension buffer may comprise an aqueous solution comprising 50 mM Tris and 10 mM EDTA at pH 8.0.

Suitable lysates or buffers may include one or more denaturants and one or more disruptive agents in an aqueous carrier medium. The denaturant may be a nucleic acid denaturant such as an alkaline salt. Suitable basic salts may include sodium hydroxide, potassium hydroxide, calcium hydroxide, and the like. Suitable lipolytic agents may include ionic surfactants. Exemplary ionic surfactants include sodium cholate, sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC), N-lauroyl sarcosinate, cetyltrimethylammonium bromide ( CTAB), bis(2-ethylhexyl)sulfosuccinate, etc. In some embodiments, the lysis buffer may be a formulation comprising 1% SDS and 200 mM sodium hydroxide.

Exemplary lysates can include NaOH containing from about 10 mM to about 500 mM, from about 50 mM to about 250 mM, or from about 100 mM to about 200 mM and up to about 10% SDS, up to about 5% SDS, or up to about 1% NaOH. Aqueous solution of SDS. The lysis buffer may contain other reagents, as will be apparent to those skilled in the art.

Suitable neutralizing solutions include, in an aqueous carrier medium, one or more agents capable of neutralizing the surfactant/alkaline solution present in the lysate. In some embodiments, the neutralizing solution may include a suitable acetate salt at a pH > 4 from about 0.5M to about 5M. An exemplary neutralizing solution may include an aqueous solution of about 3M potassium acetate, pH ~5.

Suitable wash buffers may include, from 0.1 mM to about 100 mM salt in an aqueous carrier medium, from about 0.5 mM to about 500 mM of a suitable biological buffer such that the pH of the wash buffer is at least about 6.0 or higher, and at least A suitable alcohol such as ethanol or isopropanol is 5%, at least 10%, or at least 15% by volume. Optionally, the wash buffer may include from about 0.01% by volume up to about 10% by volume of a suitable nonionic detergent, e.g., TRITON X-100, CHAPS or NP-40. In some embodiments, the addition of such detergents enhances the removal of unwanted endotoxins from the preparation. Exemplary wash buffers may include 1M NaCl, 50 mM MOPS at pH > 8.0, 15% by volume isopropanol, and 0.5% by volume TRITON X-100. In some embodiments, the wash buffer may comprise an aqueous solution at pH 5.0 containing 800 mM NaCl and 100 mM sodium acetate trihydrate. In some embodiments, the wash buffer formulation may be a formulation comprising, for example, 100 mm sodium acetate trihydrate at 1.5 NaCl, pH 5.0.

Suitable collection/elution buffers may include, up to about 50 mM of a suitable biological buffer, 5.0 < pH < 9.0, and up to about 10 mM of a suitable chelating agent in an aqueous carrier medium. Exemplary collection/elution buffers can include 10 mM Tris HCL, pH 8.0, and 1 mM EDTA.

In some embodiments, an equilibration buffer may optionally be passed freely through the solid support matrix prior to its use. The equilibration buffer may contain up to 1 M salt, up to 500 mM of a suitable biological buffer, 5.0 < pH < 9.0, up to about 10% by volume of a suitable non-ionic detergent and up to about 20% by volume of alcohol. Exemplary equilibration buffers can include, for example, 750 mM NaCl, 50 mM MOPS at pH 7, 15% by volume isopropanol, and about 0.15% by volume TRITON X-100.

In some embodiments, a precipitation buffer may optionally be allowed to pass through the solid support matrix prior to use of the solid support matrix. The precipitation buffer may contain up to 5M potassium acetate, up to 500 mM of a suitable biological buffer at 5.0<pH<9.0. An exemplary precipitation buffer can include, for example, 3.1 M potassium acetate at pH 5.5.

Also provided herein are alternative methods of purifying nucleic acids using embodiments of the bioprocessing device, comprising one or more of: selecting the number of cartridges to be used; inserting the cartridges into the bioprocessing device; inflating the bladder to further Stabilize the box; pump the cells from the sample container through the bioprocessing chamber to the waste container to trap the cells for 2100 seconds with a 700 ms pump delay between pumps; release the pressure in the box for 1 second; The pump draws RNase into the box for 4 seconds with a pump delay of 800 ms between pump strokes; by pumping resuspension buffer into the lysis buffer reservoir for 50 ms with a pump delay of 800 ms between pump strokes seconds, to mix the resuspension buffer with the lysis buffer; use the pump to pump the resuspension buffer/lysis buffer mixture back to the resuspension buffer storage pool for 80 seconds, and there is an 800ms delay between pump strokes; The resuspension/lysis buffer mixture was pumped through the bioprocessing chamber with captured cells to resuspend the cells for 150 seconds with an 800ms delay between pump strokes; preset valve 1 second; using pump with 1200ms between pump strokes Time-delay, mix lysate/resuspension buffer with cells for 100 seconds; use pump delay of 1200ms between pump strokes, pump neutralization mixture to storage containing resuspended cells and lysate/resuspension buffer Pool for 60 seconds; mix neutralization mix with resuspension/lysis buffer mix for 270 seconds using a pump delay of 2500 ms between pump strokes; remove genomic waste from system for 1 second; open a control fluidic connector , for 3 seconds; with a pump delay of 2500ms, the lysis/resuspension/neutralization buffer with cells is pumped through the second bioprocessing chamber to clarify DNA from cell debris and bind DNA in the other bioprocessing chamber Medium, for 400 seconds; then use an 800ms pump delay to wash the genome filter for 10 seconds; then preset the valve for 1 second and allow the genome filter to air dry for 2 seconds; then use a 1100ms pump delay , pump the elution buffer through the membrane for 20 seconds, then mix the eluted solution with isopropanol twice for 20 seconds using a pump delay of 800 ms; then clear the line to waste for 1 second; Then open the process valve within 3 seconds; then release the pressure in the box for 1 second; use a pump delay of 1100ms to pump the precipitated DNA mixture through another bioprocessing chamber with a PPTR membrane to capture the DNA, Last 60 seconds; then use 1100ms pump delay to pump 70% ETOH through the biological treatment chamber for 20 seconds; then remove residual ETOH to waste for 1 second; then dry the PPTR membrane for 90 seconds; then from The bioprocessing cartridge releases pressure for 1 second; then pumps the final eluate through the PPTR membrane into the collection tube using a pump delay of 3000 for 120 seconds; once the run is complete, deflates the bladder for 20 seconds seconds so that the box can be removed. The whole program runs for about 3690 seconds.

Other embodiments, apparatus, methods and cartridges are described below. In the dimensions provided below for the cartridge, height is the Y axis from top to bottom, width is the X axis from left to right, and depth is Z into the paper of the bioprocessing cartridge embodiment when viewed from shown in 13A Shaft (or minimum specification).

IV. Embodiment

Unless otherwise stated in the examples, automated western blot processing was performed using the processing lanes and valves on the upper part of the bioprocessing card. This channel is typically the channel associated with process valve 1227 in FIG. 12 .

A. Examples 1 and 2

Using the embodiment of the automated processing apparatus shown in Figures 1C and 1D and the embodiment of the bioprocessing cassette shown in Figures 12, 13A-B, and 14 to perform western blots (Figure 24A), the results were compared with the manual method (Figure 24B) as follows )Compare:

Reagents and Equipment

NuP AGE LDS sample buffer (Invitrogen cat#NP0007)

NuP AGE MES SDS running buffer (Invitrogen cat#NP0002)

NuP AGE Reducing agent (Invitrogen cat#NP0004)

See Blue Plus2 Prestained Standard (Plus2 Prestained Standard, Invitrogencat #LC5925)

NuP AGE Antioxidant (Invitrogen cat#NP0005)

Gels = NuPAGE 4-12% BT IPG Well (Invitrogen cat#NP0330BOX)

iBlot Gel Transfer Device(Invitrogen cat#IB1OO1)

iBlot Regular Transfer Stack-Mini (nitrocellulose) (Invitrogen cat#IB3010-02)

Western Breeze Chromogenic Kit - Anti-Rabbit (Invitrogen cat#WB7105)

Rabbit anti-E.coli antibody (Dako cat#B0357)

plan

1. Western blot preparation: Prepare 4 μg of E.coli lysate in NuPAGE LDS sample buffer and 5 μl of SeeBlue Plus2 standards were loaded into a NuPAGE 4-12% BT IPG well format gel and run at 200V for 34 minutes. Then use iBlot GelTransfer Device to transfer the protein on the gel to the nitrocellulose membrane (iBlot Regular Transfer Stack) according to the user manual provided with the device.

2. Preparation of immunoassay reagents: with WesternBreeze For the reagents in the chromogenic kit, prepare blocking reagent, washing buffer and primary antibody diluent according to the user manual provided with the kit. The Dako anti-E.coli primary antibody was diluted 1:1000 in the primary antibody diluent. The secondary antibody used was prepared and used included in WesternBreeze Alkaline phosphatase-conjugated goat anti-rabbit antibody in the chromogenic kit. Prepare enough reagents in one batch to process blots processed by automated instruments and blots processed by manual methods (see below).

3. Blot processing: Cut the nitrocellulose membrane (after transfer) described in part 1 of this protocol into two halves, one half is used for the immunodetection implemented in the automated instrument, and the other half is used for WesternBreeze Standard manual procedures described in the chromogenic kit user manual were performed in the Falcon dish provided with the kit.

4. Automated instrument: the following reagents are loaded into the automated instrument tray of the embodiment shown in Figure 6: blocking agent, rinse solution (water), primary antibody, washing solution (WesternBreeze Wash buffer), secondary antibody, wash solution (WesternBreeze Wash buffer - second aliquot), rinse (water - second aliquot). Insert the bioprocessing cartridge into the slot of the instrument, load half of the blot membrane into the blot holder (made of die-cut PVC film) of the embodiment shown in Figure 21A, and insert it into the instrument's bioprocessing cartridge. cavity.

5. Automated Protocol: After inserting the blotting membrane into the bioprocessing cartridge, start the protocol on the instrument, the protocol consists of the following steps, each repeat/step is performed automatically without the involvement of other people, but the protocol Each step of the process is displayed on the GUI.

a. Closed: 1 x 30 minutes - WesternBreeze sealant

b. Rinse: 2 x 5 minutes - water

c. Primary antibody: 1×60 minutes-1:1000 rabbit anti-E.coli antibody in antibody diluent

d. Washing: 4 x 5 minutes - WesternBreeze wash buffer

e. Secondary antibody: 1×30 minutes-WesternBreeze Goat anti-rabbit AP conjugate

f. Washing: 4 x 5 minutes - WesternBreeze wash buffer

g. Rinse: 3 x 2 minutes - water

For each repetition of the above steps, the times shown do not include the time to fill the chamber with the relevant reagent and represent only the recirculation time of the reagent through the chamber. At the end of each repetition of each step, the cavity is drained before starting the next repetition/step, and the drain time is also not included in the displayed time. The fill/drain time is relatively short for all steps or repetitions of the order of about 1 minute. As used herein and throughout the examples, a number followed by a time-following "x" is intended to represent the number of repetitions of the step performed within the time shown. Thus, as included above, in "Rinse: 2 x 5 minutes" means 2 repetitions of filling the cavity with water, recirculating the water for 5 minutes and draining, or in other words, filling the cavity with water, recirculating the water for 5 minutes and draining (1 repetition), the cavity was subsequently filled with water, water was recirculated for 5 minutes and drained (2nd repetition).

For each step, the manual method used the same length of time and the same number of replicates as the automated method, but each replicate/step was performed manually. The outline of the manual method is as follows: First half of the blot for manual processing was put into the WesternBreeze The blocking reagent was poured into the Falcon dish provided with the chromogenic kit, and the dish was placed on a rotating table for 30 minutes. After 30 minutes, pour off the sealer by hand and add rinse water by hand. This method of manually adding reagents, spinning/incubating for a set time, pouring and then repeating adding the next reagent is repeated for each repetition of each step listed above for the automated instrument, except they are done manually.

6. Display: After the above incubation steps are completed, the two blot halves are rinsed with water and incubated in a chromogenic substrate (from WesternBreeze Incubate for 20 minutes in the Chromogenic Kit).

The results are shown in Figures 24A and B.

B. Examples 3 and 4

Using the embodiment of the automated processing apparatus shown in Figures 1C and 1D and the embodiment of the bioprocessing cassette shown in Figures 12, 13A-B and 14 to perform western blots, the results (Figure 25A) were compared with the manual method (Figure 25B) as follows )Compare:

Reagents and Equipment

NuP AGE LDS sample buffer (Invitrogen cat#NP0007)

NuP AGE MES SDS running buffer (Invitrogen cat#NP0002)

NuP AGE Reducing agent (Invitrogen cat#NP0004)

See Blue Plus2 pre-stained standard (Invitrogen cat#LC5925)

NuP AGE Antioxidant (Invitrogen cat#NP0005)

Gels = NuPAGE 4-12% BT IPG Well (Invitrogen cat#NP0330BOX)

iBlot Gel Transfer Device(Invitrogen cat#IB1OO1)

iBlot Regular Transfer Stack-Micro(PVDF)(Invitrogen cat#IB4010-02)

BupH ^TM Phosphate Buffered Saline (Pierce cat#28372)

Surfact-AMPs 20(Pierce cat#28320)

Skimmed milk (Carnation)

Goat anti-rabbit IgG HRP conjugate (Jackson cat#111-035-003)

Rabbit anti-E.coli antibody (Dako cat#B0357)

ECL HRP Western Blotting Substrate (Pierce cat#32209)

Copier transparent film (3M PP2500)

Fuji photometer (Fuji LAS-1000)

plan

1. Western blot preparation: Prepare 4 μg of E.coli lysate in NuPAGE LDS sample buffer and 5 μl of SeeBlue Plus2 standards were loaded into NuPAGE 4-12% BT IPG well gels and run at 200V for 34 minutes. Then use iBlot Gel Transfer Device to transfer the protein on the gel to PVDF membrane (iBlot Regular Transfer Stack).

2. Preparation of immunoassay reagents:

a. PBST = Mix 2 sachets of BupH Tris Buffered Saline + 10ml Tween 20 (1 vial of Surfact-Amps 20) and dilute to IL with deionized water.

b. Blocker = Dissolve/dilute 1.25g skim milk (NFDM) to 25ml with PBST.

c. Wash buffer = PBST

d. Primary antibody solution = mix 25ml of PBST with 25μl of Dako anti-E.coli primary antibody

e. Two 2° Ab solution = mix 25ml of PBST with 5μl of Jackson HRP-coupled goat anti-rabbit IgG antibody.

3. Blot processing: the PVDF membrane (after transfer) described in part 1 of this protocol was cut in half, one half was used for immunodetection implemented on an automated instrument, and the other half was processed using the standard manual procedure described in a WesternBreeze Process in the Falcon dish provided with the immunoassay kit.

4. Automated instrument: Load reagents into the instrument tray of the embodiment shown in Figure 6, insert the bioprocessing cartridge into the instrument, and load half of the PVDF blot membrane into the blot holder of the embodiment shown in Figure 21A , and insert it into the bioprocessing chamber of the instrument's bioprocessing cartridge.

5. Automated scheme: with automated instruments and manually as described in detail in Examples 1-2 above, the following steps were implemented:

a. Blocking: 1 x 30 min - 5% NFDM in PBST

b. Rinse: 2 x 5 minutes - water

c. Primary antibody: 1×60 min-1:1000 rabbit anti-E.coli antibody in PBST

d. Wash: 4 x 5 min - PBST

e. Secondary antibody: 1×30 min-1:5000 goat anti-rabbit HRP conjugate in PBST

f. Wash: 4 x 5 minutes - PBST

g. Rinse: 3 x 2 minutes - water

6. Display: After the above incubation steps are completed, rinse the two blotted membrane halves with water and place them side by side on a plastic copier transparent film. Pipette ECL HRP substrate onto both halves and allow to incubate for 1 min. Excess substrate was discarded and another transparent sheet was placed on top of this membrane, creating an easy-to-manipulate sandwich and keeping the blot wet during imaging. The imprinted sandwich was imaged for 3 min with a Fuji LAS-1000 photometer. The results are shown in Figures 25A and 25B.

C. Examples 5-9

Four western blots were performed using the embodiment of the automated processing apparatus shown in Figures 1C and 1D and the embodiment of the bioprocessing cassette shown in Figures 12, 13A-B, and 14, and the results (Figures 26B-26E) were compared with manual method (Figure 26A) for comparison:

Reagents and Equipment

NuP AGE LDS sample buffer (Invitrogen cat#NP0007)

NuP AGE MES SDS running buffer (Invitrogen cat#NP0002)

NuP AGE Reducing agent (Invitrogen cat#NP0004)

See Blue Plus2 pre-stained standard (Invitrogen cat#LC5925)

NuP AGE Antioxidant (Invitrogen cat#NP0005)

Gels = NuPAGE 4-12% BT IPG Well (Invitrogen cat#NP0330BOX)

iBlot Gel Transfer Device(Invitrogen cat#IB1OO1)

iBlot Regular Transfer Stack-Mini (nitrocellulose) (Invitrogen cat#IB3010-02)

Western Breeze Chromogenic Kit - Anti-Rabbit (Invitrogen cat#WB7105)

Rabbit anti-E.coli antibody (Dako cat#B0357)

plan

1. Western blot preparation: Prepare 5 blots as follows: 4 μg E.coli lysate prepared in NuPAGE LDS sample buffer and 5 μl SeeBlue Plus2 standards were loaded into NuPAGE 4-12% BT IPG well gels and run at 200V for 34 minutes. Then use iBlotGel Transfer Device to transfer the protein on the gel to the nitrocellulose membrane (iBlotRegular Transfer Stack) separately.

2. Preparation of immunoassay reagents: with WesternBreeze Reagents in the kit, prepare blocking reagent, washing buffer and primary antibody diluent according to the user manual provided with the kit. The Dako anti-E.coli primary antibody was diluted 1:1000 in the primary antibody diluent. The secondary antibody used was prepared and used included in WesternBreeze Alkaline phosphatase-conjugated goat anti-rabbit antibody in the kit. In one batch, prepare enough reagents to process 5 blots - 4 with the automated instrument and 1 blot with the manual method.

3. Automated instrument: load the following 4 groups of reagents into the automated instrument tray of the embodiment shown in Figure 6: blocking agent, rinse solution (water), primary antibody, washing solution (WesternBreeze Wash buffer), secondary antibody, wash solution (WesternBreeze Wash buffer - second aliquot), rinse (water - second aliquot). Insert the 4 bioprocessing cartridges into the individual slots of the instrument. Following the embodiment of the blot holder shown in Figure 21A, the 4 blot membranes from Step 1 above were loaded into separate blot holders and inserted into the bioprocessing cartridges in the instrument. With the WesternBreeze pre-programmed in the instrument The incubation protocol used to process these blots. The instrument uses a pre-programmed WesternBreeze The protocol simultaneously processes all four blots in the instrument.

4. Automated protocol: The following steps were performed manually with automated instruments and essentially as detailed in Examples 1-2 above:

a. Closed: 1 x 30 minutes - WesternBreeze sealant

b. Rinse: 2 x 5 minutes - water

c. Primary antibody: 1×60 minutes-1:1000 rabbit anti-E.coli antibody in primary antibody diluent

d. Washing: 4 x 5 minutes - WesternBreeze wash buffer

e. Secondary antibody: 1×30 minutes-WesternBreeze Goat anti-rabbit AP conjugate

f. Washing: 4 x 5 minutes - WesternBreeze wash buffer

g. Rinse: 3 x 2 minutes - water

5. Visualization: After the above incubation steps were completed, all blotted membranes were rinsed with water and incubated in a chromogenic substrate (from WesternBreeze kit) for 20 minutes, and imaged with a Fuji photometer (3 minutes of exposure). The results are shown in Figures 26A-26E.

D. Examples 10A-B and 11A-B

Western blots were performed using the embodiment of the automated processing apparatus shown in Figures 1C and 1D and the embodiment of the bioprocessing cartridge shown in Figures 12, 13A-B, and 14, and the results (Figures 27A and 27C) were compared with the manual method as follows (Figure 27B and 27D) for comparison:

Reagents and Equipment

NuP AGE LDS sample buffer (Invitrogen cat#NP0007)

NuP AGE MES SDS running buffer (Invitrogen cat#NP0002)

NuP AGE Reducing agent (Invitrogen cat#NP0004)

See Blue Plus2 pre-stained standard (Invitrogen cat#LC5925)

NuP AGE Antioxidant (Invitrogen cat#NP0005)

Gels = NuPAGE 4-12% BT IPG Well (Invitrogen cat#NP0330BOX)

iBlot Gel Transfer Device(Invitrogen cat#IB1OO1)

iBlot Regular Transfer Stack-Mini (nitrocellulose) (Invitrogen cat#IB3010-02)

Novex ECL Chemiluminescent Substrate Kit-(Invitrogen cat#WP200005)

Rabbit anti-E.coli antibody (Dako cat#B0357)

Goat anti-rabbit IgG-HRP conjugate (Jackson Labs cat#111-035-003)

Pierce ECL (Thermo 32109)

plan

1. Western blot preparation: Prepare 4 μg E.coli lysate in NuPAGE LDS sample buffer and 3 μl SeeBlue Plus2 standards were loaded into NuPAGE 4-12% BT IPG well gels and run at 200V for 34 minutes. Then use the iBlot Gel Transfer Device to transfer the protein to the 0.2 μm nitrocellulose membrane (Figure 27A and 27B) or 0.2 μm PVDF membrane (Figure 27C and 27D) of the iBlot RegularTransfer Stacks according to the user manual instructions provided with the device .

2. Preparation of immunoassay reagents: use the following reagents:

a. Blocking agent = 5% skim milk (NFDM) in phosphate buffered saline solution containing 0.1% Tween 20 (PBST)

b. Wash buffer = PBST

c. Primary antibody solution = 1:1000 dilution of Dako anti-E.coli primary antibody in PBST

d. Two 2° Ab solution = Jackson HRP-coupled goat anti-rabbit IgG antibody diluted 1:5000 in PBST.

3. Blot processing: Cut the membrane (after transfer) described in part 1 above into two halves, each half is used for immunodetection on an automated instrument, and the other half of each sheet is processed with WesternBreeze The same reagents and standard manual procedures described in the chromogenic kit user manual are processed in the Falcon dish provided with the kit.

4. Automated instrument: load the following reagents into the automated instrument tray of the embodiment shown in Figure 6: blocking agent, rinse solution (water), primary antibody, wash solution (wash buffer), secondary antibody, wash solution (Wash Buffer - 2nd aliquot), Rinsing Solution (Water - 2nd aliquot). The individual halves of the membrane were run separately as follows: the bioprocessing cartridge was inserted into the slot of the instrument, and the blot membrane half was loaded into a blot holder (made of die-cut PVC film) as shown in Figure 21B. ) and insert it into the bioprocessing chamber of the bioprocessing cartridge of the instrument.

5. Automated Protocol: The following steps were carried out with automated instruments (FIGS. 27A and 27C) and manually (FIGS. 27B and 27D) essentially as detailed above in Examples 1-2:

a. Closed: 1×30 minutes

b. Rinsing: 2×5 minutes

c. Primary antibody: 1×60 minutes

d. Washing: 4×5 minutes

e. Secondary antibody: 1×30 minutes

f. Washing: 4×5 minutes

g. Rinsing: 3×2 minutes

6. Display: basically as described in Examples 3-4 above, the display was performed. The results are shown in Figures 27A-27D.

E. Examples 12A and 12B

Two western blots were performed using the embodiment of the automated processing apparatus shown in Figures 1C and 1D and the embodiment of the bioprocessing cassette shown in Figures 12, 13A-B and 14:

1. Western blot preparation: Two blots were prepared as follows: 4 μg E.coli lysate prepared in NuPAGE LDS sample buffer and 3 μl SeeBlue Plus2 standards were loaded into NuPAGE 4-12% BT ZOOM IPG well gels and run at 200V for 34 minutes. Then use iBlot Gel Transfer Device to transfer the protein to the 0.2 μm PVDF membrane of iBlot Regular Transfer Stacks according to the user manual provided with the device.

2. Preparation of immunoassay reagents: with WesternBreeze For the reagents in the chromogenic kit, prepare blocking reagent, washing buffer and primary antibody diluent according to the user manual provided with the kit. The Dako anti-E.coli primary antibody was diluted 1:1000 in the primary antibody diluent. The secondary antibody used was prepared and used included in WesternBreeze Alkaline phosphatase-conjugated goat anti-rabbit antibody in the chromogenic kit. Prepare enough reagents in one batch to process 2 blots.

3. Automated instrument: load the following 2 groups of reagents into the automated instrument tray of the embodiment shown in Figure 6: blocking agent, rinse solution (water), primary antibody, washing solution (WesternBreeze Wash buffer), secondary antibody, wash solution (WesternBreeze Wash buffer - second aliquot), rinse (water - second aliquot). Following the embodiment of the blot holder shown in Figure 22B, 2 blot membranes were inserted into separate blot holders and inserted into the bioprocessing chamber of the bioprocessing cartridge of the instrument.

4. Using an automated instrument essentially as detailed in Examples 1-2, two blots were processed with the following protocol:

a. Closed: 1 x 10 minutes - WesternBreeze sealant

b. Primary antibody: 1×30 min-1:1000 rabbit anti-E.coli antibody in antibody diluent

c. Washing: 2 x 1 min and 2 x 5 min - WesternBreeze wash buffer

d. Secondary antibody: 1×30 minutes-WesternBreeze Goat anti-rabbit AP conjugate

e. Washing: 2 x 5 minutes - WesternBreeze wash buffer

f. Rinse: 3 x 1 minute - water

5. Visualization: After the above incubation steps were completed, the 2 blotted membranes were rinsed with water and incubated in chemiluminescent substrate, and imaged with a Fuji photometer (3 minutes exposure). Results are shown in Figures 28A-D.

F. Examples 13A and B

Two western blots were performed using the embodiment of the automated processing apparatus shown in Figures 1C and 1D and the embodiment of the bioprocessing cassette shown in Figures 12, 13A-B and 14:

1. Western blot preparation: Prepare 2 blots as follows: 4 μg E.coli lysate prepared in NuPAGE LDS sample buffer and 5 μl SeeBlue Plus2 standards were loaded into NuPAGE 4-12% BT ZOOM IPG well gels and run at 200V for 34 minutes. Then use the method described in the NuPAGE Bi-Tris Gel instruction manual, with 10% methanol and 1:1000 diluted antioxidant to transfer the protein to a 0.45 μm nitrocellulose membrane (Example 13A) or a 0.45 μm PVDF membrane ( Example 13B) above.

2. Preparation of immunoassay reagents: use the following reagents:

a. Blocking agent = 5% skim milk (NFDM) in phosphate buffered saline solution containing 0.1% Tween 20 (PBST)

b. Wash buffer = PBST

c. Primary antibody solution = Dako anti-E.coli primary antibody diluted 1:1000 in PBST

d. Two 2° Ab solution = Jackson HRP-coupled goat anti-rabbit IgG antibody diluted 1:5000 in PBST

3. Automated instrument: Load two sets of reagents into the automated instrument tray of the embodiment shown in Figure 6, and insert 2 blot membrane cassettes into separate blot holders according to the embodiment of the blot holder shown in Figure 22B and insert it into the bioprocessing chamber of the instrument's bioprocessing cartridge.

4. Automated protocol: Using an automated instrument essentially as detailed in Examples 1-2, two blots were processed with the following protocol:

a. Closed: 1×60 minutes

b. Washing: 2×1 minute

c. Primary antibody: 1×60 minutes

d. Washing: 2 x 1 minute, 1 x 15 minutes and 2 x 5 minutes

e. Secondary antibody: 1×60 minutes

f. Washing: 2 x 1 minute, 1 x 15 minutes and 2 x 5 minutes

5. Display: basically as described in Examples 3-4 above, the display was performed. The results are shown in Figures 29A and 29B.

G. Examples 14A and B

Two western blots were performed with the embodiment of the automated processing apparatus shown in Figures 1C and 1D and the embodiment of the bioprocessing cassette shown in Figures 12, 13A-B and 14:

1. Western blot preparation: Prepare two blots as follows: 4 μg E.coli lysate prepared in NuPAGE LDS sample buffer and 3 μl SeeBlue Plus2 standards were loaded into NuPAGE 4-12% BT ZOOM IPG well gels and run at 200V for 34 minutes. Then use the iBlot Gel Transfer Device to transfer the protein to the 0.2 μm nitrocellulose membrane (Example 14A) and 0.2 μm PVDF membrane (Example 14B) of iBot Regular Transfer Stacks according to the user manual provided with the device.

2. Preparation of immunoassay reagents: with WesternBreeze For the reagents in the chromogenic kit, prepare blocking reagent, washing buffer and primary antibody diluent according to the user manual provided with the kit. The Dako anti-E.coli primary antibody was diluted 1:1000 in the primary antibody diluent. The secondary antibody used was prepared and used included in WesternBreeze Alkaline phosphatase-conjugated goat anti-rabbit antibody in the chromogenic kit. Prepare enough reagents in one batch to process 2 blots.

3. Automated instrument: load the following 2 groups of reagents into the automated instrument tray of the embodiment shown in Figure 6: blocking agent, rinse solution (water), primary antibody, washing solution (WesternBreeze Wash buffer), secondary antibody, wash solution (WesternBreeze Wash buffer - second aliquot), rinse (water - second aliquot). Following the embodiment of the blot holder shown in Figure 22B, 2 blot membranes were inserted into separate blot holders and inserted into the bioprocessing chamber of the bioprocessing cartridge of the instrument.

4. Automated scheme: use the automated instrument to process the two blots with the following scheme basically as detailed in the above examples 1-2:

a. Closed: 1 x 10 minutes - WesternBreeze sealant

b. Primary antibody: 1×30 minutes-1:1000 rabbit anti-E.coli antibody in antibody diluent

c. Washing: 2 x 1 min and 2 x 5 min - WesternBreeze wash buffer

d. Secondary antibody: 1×30 minutes-WesternBreeze Goat anti-rabbit AP conjugate

e. Washing: 2 x 5 minutes - WesternBreeze wash buffer

f. Rinse: 3 x 1 minute - water

5. Display: After the above incubation steps were completed, 2 blotted membranes were rinsed with water and incubated in chemiluminescent substrate for 5 minutes, and imaged with a Fuji photometer (3 minutes exposure). The results are shown in Figures 30A and 30B.

H. Examples 15A-B and 16A-B

The results of western blots (FIGS. 31A and 31C) performed with the embodiment of the automated processing apparatus shown in FIGS. 1C and 1D and the embodiment of the bioprocessing cassette shown in FIGS. 12, 13A-B and 14 were compared as follows Manual method (Figure 31B and 31D) for comparison:

1. Western blot preparation: Prepare two blots as follows: 4 μg E.coli lysate prepared in NuPAGE LDS sample buffer and 3 μl SeeBlue Plus2 standards were loaded into NuPAGE 4-12% BT ZOOM IPG well gels and run at 200V for 34 minutes. Then use the iBlot Gel Transfer Device to transfer the protein to the 0.2 μm nitrocellulose membrane (Figure 31C and 31D) and 0.2 μm PVDF membrane (Figure 31A and 31B) of iBot Regular Transfer Stacks according to the user manual instructions provided with the device superior.

2. Preparation of immunoassay reagents: with WesternBreeze For the reagents in the chromogenic kit, prepare blocking reagent, washing buffer and primary antibody diluent according to the user manual provided with the kit. The Dako anti-E.coli primary antibody was diluted 1:1000 in the primary antibody diluent. The secondary antibody used was prepared and used included in WesternBreeze Alkaline phosphatase-conjugated goat anti-rabbit antibody in the chromogenic kit. Prepare enough reagents in one batch to process blots processed by automated instruments and blots processed by manual methods (see below).

3. Blot processing: the membrane described in part 1 of this example (after transfer) was cut in half, and half of each sheet was used for immunoassays implemented in automated instruments, and the other half of each sheet was used for Western Breeze Standard manual procedures described in the chromogenic kit user manual are processed in the Falcon dish provided with the kit.

4. Automated instrument: For each automated process, the following reagents are loaded into the automated instrument tray of the embodiment shown in Figure 6: blocking agent, rinse solution (water), primary antibody, washing solution (Western Breeze Wash buffer), secondary antibody, wash solution (WesternBreeze Wash buffer - second aliquot), rinse (water - second aliquot). The bioprocessing cartridge is inserted into the slot of the instrument, and one half of the relevant blot membrane is loaded into the blot holder of the embodiment shown in Figure 22B and inserted into the bioprocessing chamber of the bioprocessing cartridge of the instrument.

5. Automated protocol: The following steps were performed with automated instruments (the results of which are shown in Figures 31A and 31C) and manually (the results of which are shown in Figures 31B and 31D). For automated processing, the reagents are recirculated in the bioprocessing chamber of the bioprocessing cartridge using the channel associated with process valve 1440 in FIG. 14 according to the following scheme:

a. Closed: 1×30 minutes

b. Rinsing: 2×5 minutes

c. Primary antibody: 1×60 minutes

d. Washing: 4×5 minutes

e. Secondary antibody: 1×30 minutes

f. Washing: 4×5 minutes

g. Rinsing: 3×2 minutes

6. Visualization: After completion of the above incubation step, the blot was rinsed with water and incubated in chemiluminescence substrate for 5 minutes, and imaged with a Fuji luminometer (3 minutes exposure). The results are shown in Figures 31A-31D.

I. Examples 17A-B and 18A-B

Western blots were performed as follows using the embodiment of the automated processing apparatus shown in Figures 1C and ID and the embodiment of the bioprocessing cartridge shown in Figures 12, 13A-B and 14.

1. Western blot preparation: Prepare two blots as follows: 4 μg E.coli lysate prepared in NuPAGE LDS sample buffer and 3 μl SeeBlue Plus2 standards were loaded into NuPAGE 4-12% BT ZOOM IPG well gels and run at 200V for 34 minutes. Then use the iBlot Gel Transfer Device to transfer the protein to the 0.2 μm PVDF membrane of iBot Regular Transfer Stacks according to the user manual provided with the device.

2. Blot processing: Cut the membrane (after transfer) described in part 1 of this example in half and use the NFDM/TBST reagent described in Example 13A-B (Figure 32C and 32D) or Example 10A-B and For the NFDM/PBST reagents described in 11A-B (Figs. 32A and 32B), half of each blot was used for immunoassays implemented in an automated instrument, and the results are shown in Figs. 32A and 32C, each of The other half was done manually, the results of which are shown in Figures 32B and 32D.

3. Automated instrument: For each automated process, load the reagents into the automated instrument tray of the embodiment shown in Figure 6: insert the bioprocessing cartridge into the slot of the instrument, and load half of the relevant blot membrane into the tray shown in Figure 22B. into the blot holder of the illustrated embodiment and insert it into the bioprocessing chamber of the bioprocessing cartridge of the instrument.

4. Automated Protocol: The following steps were performed with automated instruments (the results of which are shown in Figures 32C and 32D) and manually (the results of which are shown in Figures 32A and 32B). For automated processing, the reagents are recirculated in the bioprocessing chamber of the bioprocessing cartridge using the channel associated with the process valve 1440 in FIG. 14 according to the following scheme:

a. Closed: 1×30 minutes

b. Rinsing: 2×5 minutes

c. Primary antibody: 1×60 minutes

d. Washing: 4×5 minutes

e. Secondary antibody: 1×30 minutes

f. Washing: 4×5 minutes

g. Rinsing: 3×2 minutes

5. Display: basically as described in Examples 3-4, the display was carried out. The results are shown in Figures 32A-32D.

For each of Examples J-M, the following bovine serum albumin (BSA) samples (prepared individually for each example) were prepared and loaded into NuPage 4-12% BT 10 microwells for BSA blotting Microgel, and run at 200V for about 34 minutes:

5 μl Sensitive Prestained Standard (Sharp Prestained Standard, Invitrogen Cat#LC5800)

8 μl Magic Mark XP Western Protein Standard (Invitrogen Cat#LC5602)

50ng BSA (5μl of 10ng/μl BSA) (BSA-Sigma Cat#A-3059)

25ng BSA (5μl of 5ng/μl BSA)

10ng BSA (5μl of 2ng/μl BSA)

J. Examples 19A and 19B

Two western blots were performed using the embodiment of the automated processing apparatus shown in Figures 1C and 1D and the embodiment of the bioprocessing cartridge shown in Figures 12, 13A-B, and 14, and the results (Figure 33B) were compared with the manual method as follows (Figure 33A) for comparison:

1. Western blot preparation: Load and prepare a set of BSA samples as above. Then use the iBlot Gel Transfer Device to transfer the protein to the 0.2 μm nitrocellulose membrane of the iBot Regular Transfer Stacks according to the user manual provided with the device.

2. Preparation of immunoassay reagents: with WesternBreeze For the reagents in the chromogenic kit, prepare blocking reagent, washing buffer and primary antibody diluent according to the user manual provided with the kit. The Dako anti-E.coli primary antibody was diluted 1:1000 in the primary antibody diluent. The secondary antibody used was prepared and used included in WesternBreeze Alkaline phosphatase-conjugated goat anti-rabbit antibody in the chromogenic kit. Prepare enough reagents in one batch to process two blots.

3. Blot processing: the membrane (after transfer) described in part 1 of this example was cut in half, and half of each membrane was used for the immunodetection implemented in the automatic instrument (as shown in Figure 33B), each The other half of the film was stretched with WesternBreeze Standard manual procedures described in the Chromogenic Kit User Manual were processed in the Falcon dish provided with the kit (as shown in Figure 33A).

4. Automated Instruments: For automated processing, load the following reagents into the automated instrument trays of the embodiment shown in Figure 6: Blocker, Rinse (Water), Primary Antibody, Wash (WesternBreeze Wash buffer), secondary antibody, wash solution (WesternBreeze Wash buffer - second aliquot), rinse (water - second aliquot). Insert the bioprocessing cartridge into the slot of the instrument, load half of the blot membrane into the blot holder of the embodiment shown in Figure 22B, and insert it into the bioprocessing chamber of the bioprocessing cartridge of the instrument.

5. Automated scheme: Carry out the following steps according to the following scheme with automated instruments (the results are shown in Figure 33B):

a. Closed: 1×30 minutes

b. Rinsing: 2×5 minutes

c. Primary antibody: 1×60 minutes

d. Washing: 4×5 minutes

e. Secondary antibody: 1×30 minutes

f. Washing: 4×5 minutes

g. Rinsing: 3×2 minutes

6. Visualization: After the above incubation steps were completed, the 2 blotted membranes were rinsed with water and incubated in chemiluminescent substrate, and imaged with a Fuji luminometer (3 min exposure). Blots were then rinsed and incubated in chromogenic substrate for 20 minutes, rinsed, allowed to dry slightly, and imaged with an Epson 4990 scanner. Chromogenic imaging results are shown in Figures 33A and 33B. The lanes from left to right are -5μl sensitive pre-stained Marker, 8μl 1:10 diluted MagicMark, BSA (50ng, 25ng, 10ng).

K. Examples 20A and 20B

Essentially the same as in Example 19A-B, except that the protein was transferred to a 0.2 μm PVDF membrane, with the embodiment of the automated processing apparatus shown in Figures 1C and 1D and as shown in Figures 12, 13A-B and 14 Two western blots were performed with an embodiment of the bioprocessing cartridge (results shown in FIG. 34B ) and manually (results are shown in FIG. 34A ), and chromogenic imaging results are shown in FIGS. 34A and 34B . The lanes from left to right are -5μl sensitive pre-stained Marker, 8μl 1:10 diluted MagicMark, BSA (50ng, 25ng, 10ng).

L. Examples 21A and 21B

The results of two western blots ( FIG. 35B ) were compared with those obtained by manual methods using the embodiment of the automated processing apparatus shown in FIGS. 1C and 1D and the embodiment of the bioprocessing cassette shown in FIGS. 12, 13A-B, and 14. The results (Figure 35A) were compared to:

1. Western blot preparation: A set of BSA samples described above was loaded and prepared as above. Then use the iBlot Gel Transfer Device to transfer the protein to the 0.2 μm nitrocellulose membrane of the iBot Regular Transfer Stacks according to the instructions in the user manual provided with the device.

2. Imprinting treatment: cut the membrane described in Part 1 of this example (after transfer) in half, use the NFDM/TBST reagent described in Example 13A-B, and half of the imprinted membrane is used for automated instruments (as shown in Figure 35A ) and manually (as shown in Figure 35B) implemented immunoassays.

3. Automated instrumentation: For automated processing, load reagents into the automated instrument tray of the embodiment shown in Figure 6: insert the bioprocessing cartridge into the slot of the instrument, load half of the blot into the implementation as shown in Figure 22B the blot holder of the protocol and insert it into the bioprocessing chamber of the instrument's bioprocessing cartridge.

4. Automated protocol: The following steps were performed with automated instruments (the results of which are shown in Figure 35B) and manually (the results of which are shown in Figure 35A).

a. Closed: 1×60 minutes

b. Washing: 2×1 minute

c. Primary antibody: 1×60 minutes

d. Washing: 2 x 1 minute, 1 x 15 minutes and 2 x 5 minutes

e. Secondary antibody: 1×60 minutes

f. Washing: 2 x 1 minute, 1 x 15 minutes and 2 x 5 minutes

5. Visualization: After completion of the above incubation steps, 2 blots were rinsed with water and incubated in HRP chemiluminescent substrate, and imaged with a Fuji luminometer (3 min exposure). Blots were then rinsed and incubated with TMB HRP chromogenic substrate for 20 minutes, rinsed, allowed to dry slightly, and imaged with an Epson 4990 scanner. The chromogenic imaging results are shown in Figure 33. The lanes from left to right are -5μl sensitive pre-stained Marker, 8μl 1:10 diluted MagicMark, BSA (50ng, 25ng, 10ng).

M. Examples 22A and 22B

Two western blots were performed with the embodiment of the automated processing apparatus shown in FIGS. 1C and 1D and the embodiment of the bioprocessing cassette shown in FIGS. Example 19A-B The results obtained by the same manual method (FIG. 36B) were compared, except that for the automated process, the reagents were recirculated in the bioprocessing chamber of the bioprocessing cartridge using the channel associated with the process valve 1440 in FIG. 14 . The chromogenic imaging results are shown in FIG. 34 . The lanes from left to right are 5 μl sensitive pre-stained Marker, 8 μl 1:10 diluted MagicMark, BSA (50ng, 25ng, 10ng).

Unless explicitly stated in the examples, for Examples N-W, all fluids, solutions, reagents, mixtures, waste or Pumping/moving of any other fluid product of the embodiment. Furthermore, in some steps of the described embodiments, the fluid may pass through a membrane/bioprocessing chamber located in the cartridge after being drawn up into the cartridge and before being expelled from the cartridge.

N. Example 23

The results of nucleic acid purification performed using the automated processing apparatus shown in FIGS. 1A and 1B and the cassettes shown in FIGS. 16-18 are shown in FIG. 37 . The amount of genomic DNA collected with the described protocol varied with or without a flow diffuser incorporated into the device. During resuspension and lysis of cells, the amount of genomic DNA was reduced by using a flow diffuser when pumping the lysed cell mixture from the lysis buffer reservoir to the resuspension buffer/RNaseA buffer mixture reservoir. The amount of genomic DNA detected 3710 using a system incorporating a flow diffuser is less than the amount of genomic DNA detected 3720 using a system not incorporating a flow diffuser. However, the amount of plasmid DNA detected with the two devices 3730, 3740 remained comparable.

1. Cell capture: 250mL of E.coli-containing medium was pumped into the bioprocessing box from the sample storage tank located outside the bioprocessing box, and passed through the bioprocessing chamber containing the Bla065 membrane. Cells are filtered from the medium, and clarified medium passes through the cassette to waste storage. An air pressure of 20 PSI was then applied to the inlet side of the Bla065 cell capture membrane to remove residual medium from the membrane and cassette and into a waste reservoir.

2. Resuspension and lysing of cells: the RNaseA solution is pumped into the box from the external reagent storage pool, pumped out of the box into the reagent storage pool containing the resuspension buffer, and the RNaseA and the resuspension buffer are mixed together through the box. The resuspension buffer/RNaseA mixture is then drawn into the cassette, passing through the membrane, dislodging the cells trapped by the capture membrane. Cells are removed from the capture membrane and passed through the cassette into a reagent reservoir containing lysis buffer. Approximately 3/4 of the lysed cell mixture was then pumped back from the lysis buffer reservoir to the resuspension/RNaseA buffer mixture reservoir. Air pressure of 32 PSI was then applied through the outlet side of the cell capture membrane to dislodge residual captured cells into the lysis buffer reservoir. Residual captured cells are then pumped from the lysis buffer reservoir to the resuspension/RNaseA mixture reservoir.

3. Neutralization - The neutralization buffer is then pumped from the neutralization buffer reservoir to the reservoir containing the lysed cells. The lysed cells/neutralization buffer are then pumped back into the lysis buffer reservoir. The device was then paused for 3 min to allow the layer of cell debris to separate from the layer of clarified lysed phase.

4. Clarification/Binding - Cells are then decontaminated by pumping the layer of cell debris from the reservoir to the cassette, through the Extra-Thick (Xthick) fiberglass clarification membrane and the anion exchange DNA binding membrane, and pumping out of the cassette into the waste storage reservoir. Pieces clarified. Anion exchange wash buffer is then pumped from the anion exchange wash buffer reservoir into the cartridge, through the membrane, and out to waste reservoir. An air pressure of 32 PSI was then applied through the anion exchange membrane to ensure that all waste was collected from the membrane into the waste container. Anion exchange elution buffer is then pumped from the anion exchange elution buffer into the cartridge, across the membrane, and out of the bioprocessing cartridge into a reagent reservoir containing isopropanol to precipitate pDNA. The mixture was then mixed by pumping the elution buffer/isopropanol buffer back into the anion exchange elution buffer reservoir. The mixture was then mixed again by pumping the elution buffer/isopropanol buffer from the anion exchange elution buffer reservoir back to the isopropanol reservoir. The elution buffer/isopropanol mixture was then passed through a PPTR membrane (Bla065) to capture the precipitated pDNA. The remainder of the mixture is then pumped through the cassette and into waste storage. 70% ETOH is then pumped from the ETOH reagent reservoir into the cartridge, through the PPTR membrane, and out to the waste reservoir. Air at 32 PSI was then passed through the membrane for 1.5 minutes to air dry the membrane and allow any waste to pass through the membrane to be collected into a waste storage tank. The final TE elution buffer is then pumped from the TE elution buffer reservoir through the PPTR membrane and out to the collection tube. As shown in Figure 37, the amount of genomic DNA detected with the device with a diffuser was compared to the amount of genomic DNA detected with the device without a diffuser.

O. Example 24

Nucleic acids can be purified and captured using the bioprocessing system described in Figures 1A and 1B and the protocol described below. In contrast to Example 23, Example 24 included a partial flow diffused pump valve stem and lysis and resuspension buffers that had been premixed together prior to cell resuspension.

1. Cell capture - 250 mL of E. Coli-containing culture solution was pumped into the bioprocessing box from a disposable cell liner reservoir located outside the bioprocessing box, and passed through the bioprocessing chamber containing the Bla065 membrane. Cells are filtered from the culture fluid, and the clarified culture fluid is passed through the cassette to a waste reservoir.

2. Resuspension and lysis of cells - RNaseA solution is drawn from the reagent reservoir into the cartridge, passes through the cartridge and into the reagent reservoir containing the resuspension buffer. Lysis buffer is then pumped from the lysis buffer reservoir to the reservoir containing resuspension buffer and RNaseA. The lysed/resuspended/RNaseA mixture is then pumped into the cartridge and passed through the membrane to remove and lyse the cells from the cell capture membrane before collection into the external reservoir. The remaining cells are then pumped externally into a second reservoir.

3. Neutralization - Pump the neutralization buffer from the neutralization buffer reservoir to a separate reservoir. Cells from the second reservoir described above are then pumped into the reservoir containing neutralization buffer. Pause the automated system for 3 min to allow the cell debris phase to separate from the clarified lysate phase.

4. Clarification/Binding - The neutralized cell material is then clarified from the reservoir by pumping the diffuser pump through the Extra-Thick fiberglass clarification membrane and the anion exchange DNA binding membrane and out into the waste reservoir. Anion exchange wash buffer is then pumped from the anion exchange wash buffer reservoir through the membrane and out to waste reservoir. Air was then applied at 32 PSI, passed through the anion exchange membrane, and out to waste storage. Anion exchange elution buffer is then pumped from the anion exchange elution buffer through the membrane and out to a reservoir containing isopropanol where pDNA is precipitated. The mixture is then mixed by moving the elution buffer and isopropanol mixture from the isopropanol reservoir to the second reservoir and back to the isopropanol reservoir through the cartridge. The elution buffer/isopropanol mixture containing the precipitated pDNA is then pumped through the bioprocessing chamber containing the PPTR membrane to capture the DNA. The remainder of the medium was removed to waste. The 70% ETOH is then pumped from the ETOH storage tank through the PPTR membrane and out to the waste storage tank. The membrane was then air dried with 32 PSI air for 1.5 minutes and any material released from the PPTR membrane was collected in waste storage. The final TE elution buffer is then pumped from the TE elution buffer reservoir through the PPTR membrane and collected in a collection tube. The collected DNA is shown in Figure 38. The amount of genomic DNA detected 3810 and the amount of plasmid DNA 3820 are displayed.

P. Example 25

Nucleic acids can be purified and captured using the automated processing system shown in Figures 1A and 1B and the bioprocessing cartridges shown in Figures 16-18. Wherein the automated system is configured with variable pump speed and step timing.

1. Cell capture - 250mL of E.coli-containing culture fluid was pumped into the bioprocessing box from the disposable cell liner storage tank located outside the bioprocessing box, and passed through the bioprocessing chamber containing the Bla065 membrane. Cells are filtered from the culture fluid, and the clarified culture fluid is passed through the cassette to a waste reservoir. The system was run for a capture time of 21 minutes with an 800 ms pump delay between each stroke.

2. Resuspend and lyse cells - Pump the RNaseA solution from the RNaseA solution reservoir into the resuspension buffer reservoir for 5 seconds with a pump delay of 800 ms between each pump stroke. The resuspension buffer and RNaseA were then pumped back to the RNaseA solution reservoir and then back to the resuspension buffer reservoir for 2 seconds with a pump delay of 800 ms between each pump stroke. The resuspension/RnaseA buffer was then pumped through the cassette into the lysis buffer reservoir for 1 min with a pump delay of 800 ms between pump strokes. The RNaseA/Resuspension/Lysis Buffer is then transported from the Lysis Buffer Reservoir through the card into the Resuspension/RNaseA Buffer Reservoir with a pump delay of 800 ms between each pump stroke, allowing the mixture to mix for 1 min . The lyse/resuspension/RNaseA mixture is then pumped through the membrane where cells trapped on the cell capture membrane are dislodged from the membrane and lysed before being transported to the lysis/resuspension/RNaseA reagent reservoir. Pumping was performed for 1 minute and 45 seconds with a pump delay of 800 ms between each pump stroke. The lysis mixture with lysed cells was then pumped to the lysis buffer reagent reservoir for 1 minute and 45 seconds with a pump delay of 1100 ms between pump strokes.

3. Neutralization - Pump the lysed cells from the Lysis Buffer Reagent Reservoir to the Neutralization Buffer Reagent Reservoir with a pump delay of 1100 ms between each pump stroke for 1 minute and 45 seconds. The solution was pumped from the neutralization reservoir to the lysis mixture reservoir using a diffuser for 4.5 minutes with a pump delay of 2500 ms between pump strokes. The automated system was then paused for 3 min to allow the cell debris phase to separate from the clarified lysate phase.

4. Clarification/Binding - Cell debris and clarified lysate phase are clarified by pumping the cell debris and clarified lysate phase through the Extra-Thick glass fiber clarification membrane and then through the anion exchange DNA binding membrane to the waste reservoir with a diffuser pump for 5 min 30 seconds, the pump has a pump delay of 2500 ms between each pump stroke. Anion exchange wash buffer was then pumped from the anion exchange wash buffer reservoir through the anion exchange membrane and out to waste reservoir for 30 seconds with a pump delay of 1100 ms between each pump stroke. An air pressure of 32 PSI is then applied across the anion exchange membrane to pass the debris from the membrane into the waste storage tank. The air pressure was applied for 2 seconds before stopping and the anion exchange elution buffer was pumped through the membrane and out to the isopropanol reagent reservoir for 30 seconds with a pump delay of 1100 ms between each pump stroke. The elution buffer and isopropanol buffer were then pumped to the second reservoir and back to the isopropanol buffer reservoir, allowing the mixture to mix for 45 seconds with an interval of 1100 ms between each pump stroke. Pump delay. The waste line is then purged with 32 PSI air pressure, with any waste in the waste line passing through to the waste storage pool. Stop after 2 seconds of air pressure, open the valve to release the pressure. The system was then paused for 1 minute to allow precipitation to occur. The elution buffer/isopropanol mixture containing the precipitated pDNA was then pumped through a PPTR membrane (Bla065) to capture the DNA, and the remaining mixture was passed through to the waste reservoir for 1 min with intervals between pump strokes. 1100ms pump delay. 70% of the ETOH was then pumped from the ETOH reservoir into the cartridge, through the PPTR membrane, and out to waste for 25 seconds with a pump delay of 1100 ms between each pump stroke. Residual ETOH was removed from the line by applying an air pressure of 32 PSI to the membrane. Pause the system for 1 second. The membrane was then air dried for 1.5 minutes by applying 32 PSI air pressure through the check valve and pumping to waste. The pDNA was then eluted into the collection tube by pumping TE elution buffer through the PPTR for 5 minutes with a 2000 ms pump delay between pump strokes. The amount of genomic DNA (gDNA) detected 3910, 3920 and the amount of plasmid DNA (pDNA) detected 3930, 3940 is shown in FIG.

Q. Example 26

Nucleic acids can be purified and captured using the automated processing system shown in Figures 1A and IB and the bioprocessing cartridges shown in Figures 16-18, where the automated system is configured with varying pump speeds and step timing. Nucleic acid purification is performed using a partial flow diffuse pumping step in which applied air pressure, premixing of lysis buffer and resuspension buffer is eliminated. This example also uses the reagent reservoir tray shown in Figures 8B-8F. This protocol version ultimately removes genomic DNA (gDNA) contamination.

1. Capturing Cells - 125 mL of E. coli containing medium was drawn from the disposable cell liner reservoir into the cassette and passed through the Bla065 membrane of one of the cassette's bioprocessing chambers to filter the cells from the medium. Clarified medium was pumped out of the cassette into waste storage with a capture time of 15 minutes with a 700 ms pump delay between pump strokes. Then release the pressure and pause the system for 1 second.

2. Cell resuspension and lysis - pump the RNaseA solution from the RNaseA reagent reservoir through the cassette into the resuspension buffer reservoir for 4 seconds with an 800ms pump delay between each pump stroke. The resuspension buffer/RNaseA buffer was then pumped into the lysis buffer reservoir for 45 seconds with an 800 ms pump delay between pump strokes. The lysate/resuspension/RNaseA mixture is then drawn into the cassette and passed through the cell capture membrane. The captured cells were then removed from the membrane and lysed, and the lysed cells were pumped into the resuspension/RNaseA buffer mixture reservoir for 1 min 20 sec with an 800 ms pump delay between pump strokes.

3. Neutralization - Pump the lysed cell mixture to the second reservoir and back to the original reservoir using a diffuser for 3.5 minutes with a 2500 ms pump delay between pump strokes. Purge the waste line by blowing 32 PSI air pressure in the box for 2 seconds using the check valve. Pause the automated system for 30 s to allow the cell debris phase to separate from the clarified lysate phase.

4. Clarification/Binding - Cell debris is pumped with a diffuser pump through the Extra-Thick fiberglass clarification membrane in one bioprocessing chamber and through the anion exchange DNA binding membrane in the other bioprocessing chamber and finally to waste Reservoir to allow clarification of cell debris for 8.5 minutes with a 2500 ms delay between pump strokes. The remaining debris and buffer were then pumped to waste for 30 seconds with a 2500 ms delay between pump strokes. The remaining mixture in the lysis buffer reservoir was pumped to the waste container for 30 seconds with a 2500 ms delay between pump strokes. The anion exchange wash buffer is then pumped from the anion exchange wash buffer reagent reservoir through the anion exchange membrane to remove unwanted material pumped out to the waste reservoir for 15 seconds with an 800 ms delay between each pump stroke . Anion exchange elution buffer was then pumped through the membrane to elute captured DNA material into the isopropanol reagent reservoir for 30 seconds with an 1100 ms delay between pump strokes. The elution buffer and isopropanol buffer mixture was then pumped to the second reagent reservoir and back to the isopropanol reagent reservoir, allowing the mixture to mix for 30 seconds with an 800 ms delay between pump strokes. hour. The waste line is then purged with 32 PSI air pressure, thereby removing the waste to the waste storage tank, for 1 second. The pressure is then released by opening several valves along the waste line. The system was then paused for 2 minutes to allow the pDNA in the mixture to precipitate. The elution buffer/isopropanol mixture with the precipitated pDNA was then pumped through the PPTR membrane (Bla065) to capture the precipitated DNA while allowing the remainder of the mixture to flow to waste for 1 min, after the pump flushed There is a delay of 1100ms between. 70% ETOH was then pumped from the ETOH reagent reservoir through the PPTR membrane and out to waste. 32 PSI was then applied through the line for 1 second to remove the remaining ETOH from the line and to waste. The membrane was then air dried by applying 32 PSI air through the check valve through the membrane and into the waste storage tank. The final TE elution buffer was then applied through the PPTR membrane with a delay of 3000 ms to elute the precipitated pDNA for 1 min 20 sec. The amount of plasmid DNA detected 4010 is shown in FIG. 40 .

R. Example 27

Protein expression can be identified using the bioprocessing systems shown in Figures 1C and ID and the bioprocessing cassettes shown in Figures 12-14. In some embodiments, a nitrocellulose membrane, such as a Hybond nitrocellulose membrane, can be used with the system, although any suitable membrane can be used with the system.

1. Sample: The 3T3-L1 primordial cell line was differentiated into adipocytes. Insulin was then added to some samples to stimulate pAKT expression. Each sample contained 10 μg of differentiated 3T3-L1 adipocyte lysate.

2. Protocol: Block samples in a blocking agent such as SeaBlock/TBS/0.5% Tween 20 for 60 minutes. Any suitable blocking agent may be used including MILK, BSA, other sera, IVG. The samples are then washed 2 times for 1 minute with a wash buffer such as TBS/0.05% Tween 20. Any suitable wash buffer may be used including PBS. In addition, the percentages of components in the wash buffer can vary. Samples were then co-incubated with 1/1000 AKT (rabbit) and pAKT (mouse) primary antibodies or Glut-4 (rabbit) and GADPH (mouse) primary antibodies in SeaBlock/TBS/0.5% Tween 20 blocking agent for at least 900 minutes or overnight. Samples were then washed 3 times for 5 minutes in TBS/0.05% Tween 20 wash buffer. Then the sample was mixed with 1 nM GAR-QDaot 625 and GAM-Qdot 800 secondary antibody conjugate (secondconjugate) or 1 μg/mL GAR-AlexaFluor 790 (AF790) and GAM-AlexaFluor680 (AF680) secondary antibody conjugate in 0.01% SDS Co-incubate for 60 minutes with the blocking agent. Any suitable label that can be used includes quantum dot (Qdot) nanocrystals, including QDots 625, 605, 655, 565, 585, 705, 800, and 525, and microspheres. In some embodiments, the primary antibody conjugate can be goat anti-mouse IgG (GAM), goat anti-rabbit IgG (GAR), or streptavidin. In some embodiments, the sample can be coupled to the Click-iT Labeling/Imaging Kit. In some embodiments, the secondary antibody conjugate can be GAM, GAR, or streptavidin. Samples were then washed 3 times for 5 minutes in TBS/0.05% Tween20 wash buffer. The samples were then rinsed 2 times in water for 5 minutes.

3. Result: Q-Dot will be used Results for nanocrystals and devices described herein are shown in Figures 41A-41F, Figures 42A-42C, and Figures 43A-43F. Figures 41A-41F show the results obtained using the device shown in Figures 1C and ID. Figures 41A-41C show the results obtained with gels prepared on the bench, while Figures 41D-41F show the results obtained with the device described herein. Figures 41A and 41D show running two gels to identify the presence of AKT (rabbit) labeled with GAR-Qdot 625, Figures 41B and 41E show running two gels to detect the presence of pAKT (mouse)+GAM-Qdot 800, and Figures 41C and 41F illustrate the combined gel results shown in Figures 41A and 41B and Figures 41D and 41E, respectively. Arrows indicate lanes where both AKT and pAKT were detected.

Figures 42A-42C show the results obtained with the apparatus described in Figures 1C and ID. Figure 42A shows the presence of AKT (rabbit), where AKT has been labeled with GAR-AlexaFluor 790. Figure 42B shows the presence of pAKT (mouse), where pAKT has been labeled with GAM-AF680. Figure 42C shows an image of the gel shown in Figures 42A and 42B. Arrows indicate lanes where both AKT and pAKT were detected.

Figures 43A-43F show that different proteins are present in adipocytes, and more specifically, there are equal amounts of proteins passing through the gel. The results of Figures 43A-43F can be used to compare how much sample was loaded in each well of the gel with Qdots or AlexaFlour dye. Figure 43A shows the presence of Glut-4 (rabbit) labeled with GAR-Qdot 625. Figure 43B shows the presence of GADPH (mouse) labeled with GAM-Qdot 800. Figure 43C shows the combined image of Figures 43A and 43B. Figure 43D shows the presence of Glut-4 (rabbit) labeled with GAR-AF 790. Figure 43E shows the presence of GAPDH (mouse) labeled with GAM-AF680. Figure 43F shows the merged image of Figures 43D and 43E.

S. Example 28

Food safety analysis can be performed using the biological processing system shown in Figures 16-18. Food contamination by pathogenic microorganisms is one of the major concerns of the food industry. Traditional culture methods for food safety pathogen detection are time consuming and can take more than 24 hours from start to finish. A current need in food safety inspection is a rapid test to identify small numbers of bacteria, typically within 8 hours, in 1 cubic foot per 25 gram food sample. These needs are challenged by the rapid detection of slow-growing bacteria, since after a short (up to 6 hours) pre-enrichment step, small numbers of bacteria may be present in the culture. Detection and identification of bacterial pathogens in foods can be performed using DNA analysis, however, extraction, purification and recovery of sufficient quantities of bacterial DNA from large sample volumes are important factors in these analyses. Due to the relatively small amount of pathogenic bacteria present in the culture (typically 0.1-10 cfu/mL) even after 8 hours of enrichment, it was necessary to recover sufficient amounts of bacterial DNA for successful downstream PCR. Insufficient DNA target in the PCR reaction can result in high Ct values or no positive signal. Traditional concentration techniques include techniques such as centrifugation of large sample volumes (1 ml or more), co-precipitation of bacteria, food sample particles and other inhibitors often present in culture. Therefore, a robust DNA extraction protocol must be used before PCR or other molecular techniques can be used. However, centrifugation of large volumes of cultures enriched with pathogenic bacteria risks damaging the containers and thus seriously contaminating the equipment and work area. Using the devices provided herein in Figures 1A and 1B and the bioprocessing cartridges shown in Figures 16-18, DNA can be prepared and extracted from large sample volumes using automated methods and devices.

1. Sample preparation: Food matrix culture (10-50 mL) was applied to the pre-filter (P). The pre-filter retains large sized particles and allows bacteria to flow through the filter. The flow through, which contains most of the bacteria, is then directed to a second filter where the bacteria are captured by the filter and the flow through is discarded to waste. Bacteria captured by the second filter are then lysed on the second filter with a lysate such as PrepSeq lysate, and the lysate is directed to a silica membrane. DNA from lysed bacteria was captured by a silica membrane, and the flow-through was discarded to waste storage. The silica membrane is then washed with PrepSeq Wash Buffer to remove PCR inhibitors.

2. DNA Collection: An elution buffer such as PrepSeq Elution Buffer is pumped through the silica membrane to elute the DNA from the silica membrane. The amount of DNA eluted from the membrane can range from 40 μL to 115 μL.

3. Results: The use of the bioprocessing device herein allows the processing of very large volumes (>10 mL) of complex sample cultures in a relatively short period of time. Automated sample handling steps allow removal of food particles from the sample matrix and capture of bacterial DNA extracts with a silica membrane.

T. Example 29

Nucleic acids can be purified and captured using the bioprocessing system described in FIGS. 1A and 1B and the bioprocessing cartridges shown in FIGS. 16-18 , wherein the automated system is configured with variable pump speed and step timing. Nucleic acid purification using a partial flow diffuse pumping step in which applied air pressure, premixing of lysis buffer and resuspension buffer is removed. This example also uses the reagent reservoir tray shown in Figures 8B-8F. This protocol version optimizes the removal of genomic DNA (gDNA) contamination.

1. Cell capture - 150mL of culture fluid containing E.Coli is pumped into the bioprocessing box from the sample storage pool (disposable cell liner storage pool) located outside the bioprocessing box, and passed through the bioprocessing chamber containing the Bla065 membrane, to filter the cells from the culture medium. The clarified medium, separated from the cells, is pumped out of the cassette into a waste reservoir. Using a pump delay of 700 ms, the sample is pumped through the bioprocessing chamber for a capture time of 15 minutes, releasing the pressure held in the cartridge for 1 second.

2. Cell resuspension and lysis: the RNaseA solution is drawn into the cartridge from the external reagent reservoir, and then pumped out of the cartridge into the reagent reservoir containing the resuspension buffer. With a pump delay of 800ms, the RNaseA solution was pumped for 4 seconds. The resuspension/RNaseA buffer mixture was then mixed together. The resuspension gel/RNaseA buffer mixture is then pumped from the buffer reservoir to the lysis buffer reservoir. With a pump delay of 800ms, the mixture was pumped for 45 seconds. The lysis buffer/resuspension/RNaseA mixture is then pumped from the lysis buffer reservoir through the membrane side outlet to simultaneously remove and lyse the cells from the cell capture membrane. The mixture is pumped through the membrane side outlet, eliminating the need to use air to dislodge remaining captured cells. With a pump delay of 800 ms, the mixture was pumped for 1 minute and 20 seconds. With a pump delay of 2500 ms, the diffused lysis buffer/resuspension/RNaseA mixture was pumped to the resuspension/RNaseA mixture reservoir for 3 min 30 sec.

3. Neutralization: The lysed cells were then pumped from the first reservoir to the second reservoir for 3 minutes and 30 seconds with a pump delay of 2500 ms between each pump stroke. The mixture was then diffusion pumped from the second reservoir to the third reservoir for 6 minutes and 40 seconds with a pump delay of 2500 ms between each pump stroke. The mixture was then diffusion pumped from the third reservoir back to the second reservoir for 5 minutes with a pump delay of 2500 ms between each pump stroke. The remainder of the mixture in the third reservoir was then diffusion pumped from the third reservoir back to the second reservoir for 5 minutes with a pump delay of 2500 ms between each pump stroke. Then apply 32 PSI air with the check valve and purge the waste line for 2 seconds. The device was then paused for 5 minutes to allow cell debris and clarified lysate to phase separate.

4. Clarification/anion exchange binding: The cell debris and clarified lysate phase material is then pumped through the Extra-Thick glass fiber clarification membrane with a diffuser pump, then through the anion exchange DNA binding membrane, and pumped to the waste storage tank, so that Cell debris was clarified for 10 minutes and 30 seconds with a pump delay of 2500 ms used. Residual debris and buffer were removed with a pump delay of 2500 ms for 1 min. The anion exchange wash buffer was then pumped through the anion exchange membrane and out to waste reservoir for 40 seconds, using a pump delay of 2500 ms.

5. Anion-exchange elution and precipitation: Anion-exchange elution buffer is pumped from the buffer reservoir through the anion-exchange membrane and pumped out to the reservoir containing isopropanol for 1 minute and 30 seconds. The pump used The delay is 1100ms. Then by pumping the elution buffer and isopropanol buffer mixture from the isopropyl reservoir to the elution buffer reservoir for 20 seconds with a pump delay of 800 ms, the mixture was then pumped back to the isopropyl reservoir. Propanol reservoir for 30 seconds with a pump delay of 800 ms to allow the mixture to mix. Then purge the waste line to waste with 32 PSI air pressure for 1 second. Then release the pressure by opening the valve. The system was then paused for 2 minutes to allow precipitation to occur.

6. Capture pDNA on the Precipitator Membrane: The elution buffer/IPA mixture containing the precipitated pDNA is diffusely pumped through the PPTR membrane (Bla065) to capture the DNA. Filtered buffer was pumped to waste for 4 minutes with a pump delay of 2500ms used. 70% ETOH was then diffusion pumped from the ETOH reservoir through the PPTR membrane and out to waste for 30 seconds with a 2500 ms delay between pump strokes. Residual ETOH was removed from the line by applying air pressure of 32 PSI for 1 second, through the valve to waste. The membrane was then air dried by applying 32 PSI of air through the check valve and to waste for 1.5 minutes.

7. Final elution into the collection tube: Then with a pump delay of 3000 ms between pump strokes, apply TE elution buffer through the PPTR membrane to elute the pDNA into the collection tube for 5 minutes.

U. Example 30

The protocol described above in Example 29 was used to examine cell clumping and the effect of adding NaCl to alleviate cell clumping. The protocol was run with the bioprocessing system described in Figures 1A and 1B and the bioprocessing cassette shown in Figures 16-18.

Using the protocol described above in Example 29, 250 mM NaCl was added to 125 mL of cells on the reagent tray. Prepare an overnight culture by adding 50 uL of TOP10/DH10B-T1R glycerol stock solution to 1 L of LB broth containing 100 μg/ml ampicillin. Pour 125 mL of the bacterial culture into the reagent reservoir. Before starting a run with the device, NaCl (5M) was added to the reservoir to achieve the desired NaCl concentration.

Before capturing DH10B-T1R cells, the effect of treating cells with 0.2M NaCl solution and 0.5M NaCl solution was compared, and the comparison results are shown in Figure 44A. As shown in Figure 44A, there was a small increase in plasmid yield when using a solution of 0.2M NaCl when compared to the control, while the 0.5M NaCl solution appeared to have an adverse effect on plasmid yield.

Before capturing Top 10 cells, the effect of treating cells with 250mM NaCl solution and 300mM NaCl solution was compared, and the comparison results are shown in Figure 44B. As shown in Figure 44B, compared with the control, both NaCl concentrations increased the plasmid yield obtained from Top 10 cells, when using 250mM NaCl concentration, there was a 55% increase in the captured plasmid yield, and when using 300mM There was a 40% increase in captured plasmid yield at NaCl concentration.

V. Example 31

The protocol described in Example 29 above was used to examine cell aggregation and the effect of adding NaCl to alleviate cell aggregation. The protocol was run using the bioprocessing system described in Figures 1C and ID and the bioprocessing cartridge shown in Figures 16-18.

Pretreatment of cells with NaCl: Add 6.5 mL of 5M NaCl to 125 mL of culture in the reagent tray. The plasmid yield from cells pretreated without salt (control) was compared to that obtained from cells pretreated with 250 mM NaCl.

Figure 45A shows the plasmid yield obtained from low optical density (OD) TOP 10 cells (cells with an average density of 1.85) and high optical density TOP 10 cells (cells with an average density of 2.4-2.5), compared to those obtained from TOP 10 controls ( No salt treatment) compared with the plasmid yield. As shown in Figure 45A, the yield of plasmid collected from all TOP 10 cells treated with 250 mM NaCl was increased compared to the control. As shown in Figure 45B, the final sample volume collected after the run was not affected by salt pretreatment.

W. Example 32

The protocol described in Example 29 above was used to examine cell aggregation and the effect of adding NaCl to alleviate cell aggregation. The protocol was run with the bioprocessing system described in Figures 1C and ID and the bioprocessing cassette shown in Figures 16-18.

Pretreatment of cells with NaCl: Add 6.5 mL of 5M NaCl to 125 mL of culture in the reagent tray. The plasmid yield from cells pretreated without salt (control) was compared to that obtained from cells pretreated with 250 mM NaCl.

Figure 46 shows the plasmid yield obtained from DH10B-T1R cells pretreated with 250 mM NaCl, compared to cells pretreated without salt. As shown in Figure 46, the plasmid yield from DH10B-T1R cells was increased in cells pretreated with 250 mM NaCl compared to control cells.

The terms and expressions used herein are used as terms of description rather than limitation, and the use of such terms and expressions is not intended to exclude any equivalents or parts thereof of the features shown and described, but it is to be understood that in the present invention, Various modifications are possible within the claimed scope. Therefore, it should be understood that although the invention has been disclosed to some extent by way of preferred embodiments, exemplary embodiments and optional features, modifications and variations of the concepts disclosed herein may resort to this those skilled in the art, and such modifications and changes are considered to be within the scope of the present invention as defined in the appended claims. The specific embodiments provided herein are examples of useful embodiments of the invention, and it will be apparent to those skilled in the art that the invention may be practiced using numerous variations of the devices, device components, and method steps shown in the specification.

All references cited in this application are incorporated by reference in their entirety to the extent they are not inconsistent with the disclosure of this application. It will be apparent to those skilled in the art that methods, devices, device elements, materials, procedures, and techniques other than those specifically described herein may be employed in the practice of the present invention as broadly disclosed herein without resort to different Appropriate test methods. All art-known functional equivalents to the methods, devices, device elements, materials, procedures, and techniques specifically described herein are intended to be encompassed within the present invention.

When a group of materials, components, assemblies or compounds are disclosed herein, it is understood that all individual members of that group and all subgroups thereof are individually disclosed. When a Markush group or other subgrouping is used herein, all individual members of that group and all possible combinations and subcombinations within that group are intended to be individually encompassed by the present disclosure. Every mode or combination of components described and exemplified herein, unless otherwise indicated, can be used to practice the invention. Whenever a range is provided in the specification, for example, a temperature range, a time range, or a combination of ranges, all intermediate ranges and subranges, as well as individual numerical values, including within the given range are intended to be encompassed in the disclosure.

Claims (30)

1. A bioprocessing box comprising: a) at least one bioprocessing chamber configured to accommodate a solid support; b) a plurality of mesoscale and/or microscale process fluid channels in fluid communication with the bioprocessing chamber via at least one pump, wherein the at least one pump is included in or on the bioprocessing cartridge; c) a film or foil layer encasing the at least one bioprocessing chamber, the plurality of mesoscale and/or microscale fluid channels and the at least one pump; and d) A cartridge alignment guide configured to retain the bioprocessing cartridge in a cartridge holder of a bioprocessing device. 2. The cassette of claim 1, wherein the cassette includes at least one inlet valve located in a defined flow path of at least one of the plurality of process fluid channels. 3. The cartridge of claim 2, wherein each of said at least one inlet valve is located in a flow path between a process fluid connector and at least one of said plurality of process fluid channels, each process Fluid connectors are configured to fluidly connect the cartridge to one or more fluid containers. 4. The cartridge of claim 3, wherein the cartridge includes more than one inlet valve, wherein each inlet valve is placed in the flow path between a separate process fluid connector and one of the plurality of process fluid channels , each individual process fluid connector is configured to fluidly connect the cartridge to one or more fluid containers. 5. The cartridge of claim 3, wherein the process fluid connector is configured to connect to the fluid container through a manifold. 6. The cartridge of claim 3, wherein the process fluid connector is configured to connect to the fluid container through a suction tube and/or a withdrawal tube. 7. The cartridge of claim 3, wherein the process fluid connectors are suction and/or extraction tubes. 8. The cassette of claim 2, wherein the cassette includes an inlet valve in each flow path between each of the process fluid connectors and each of the plurality of process fluid channels. 9. The cassette of claim 1, further comprising at least one process valve located in the flow path between the pump and the chamber. 10. The cartridge of claim 1, wherein the cartridge comprises two or more bioprocessing chambers. 11. The cartridge of claim 1, further comprising a plurality of control fluid channels. 12. The cartridge of claim 11 , further comprising a control fluid connector connected to each of the plurality of control fluid channels, each control fluid connector being configured to fluidly connect the cartridge to a or multiple automated control systems. 13. The cartridge of claim 1, wherein the cartridge includes a dye chamber in fluid communication with at least one process fluid channel, wherein a material located in the dye chamber changes color when in contact with a fluid. 14. An automated biological processing unit comprising: a) one or more cartridge slots, each slot configured to accommodate a bioprocessing cartridge; b) a removable fluid container tray comprising at least one fluid container holder configured to hold containers used during bioprocessing; and c) an automated control system configured to control at least one parameter associated with bioprocessing in one or more bioprocessing cartridges, Wherein said bioprocessing box comprises: i) at least one bioprocessing chamber configured to accommodate a solid support; ii) a plurality of mesoscale and/or microscale process fluid channels in fluid communication with the bioprocessing chamber via at least one pump, wherein the at least one pump is included in or on the bioprocessing cartridge; iii) a film or foil layer encasing the at least one bioprocessing chamber, the plurality of mesoscale and/or microscale fluid channels and the at least one pump; and iv) A cartridge alignment guide configured to retain the bioprocessing cartridge in a cartridge holder of a bioprocessing device. 15. The device of claim 14, wherein the device comprises 2-8 cassette slots. 16. The apparatus of claim 14, wherein the cartridge slot further comprises a fluid manifold configured to connect one or more fluid containers in the fluid container holder to one or more process fluid connectors Fluid connections, and/or fluid manifolds, configured to connect one or more control fluid connectors to one or more automated control systems. 17. Apparatus as claimed in claim 14, wherein said cartridge slot comprises a plurality of suction and/or withdrawal tubes for accommodating the cartridge and guiding said suction and/or withdrawal tubes into said fluid container holding An opening or guide in a fluid container within the container. 18. The apparatus of claim 14, wherein said automated control system independently provides control of said pumps and said valves on bioprocessing cartridges in each of said cartridge slots. 19. An automated bioprocessing method comprising: a) providing a bioprocessing cartridge comprising i) at least one bioprocessing chamber containing a solid support therein; ii) a plurality of mesoscale and/or microscale process fluid channels in fluid communication with said biological processing chamber; iii) a film or foil layer encasing the at least one bioprocessing chamber, the plurality of mesoscale and/or microscale fluid channels and at least one pump; and iv) a cartridge alignment guide configured to retain the bioprocessing cartridge in a cartridge holder of a bioprocessing device; and b) pumping at least one process fluid through at least one of the plurality of process fluid channels and into the bioprocessing chamber. 20. The method of claim 19, wherein the pumping comprises pumping one or more reagents and/or samples into the processing chamber and into contact with the solid support. 21. The method of claim 20, wherein said pumping includes circulating at least one of said reagents from a channel near the upper portion of the chamber through a pump on the cartridge and into a channel near the bottom of the chamber. 22. The method of claim 19, wherein the pumping includes pumping at least one process fluid through or across the surface of a filter or membrane in the at least one bioprocessing chamber. 23. The method of claim 19, wherein the method comprises an automated western blot processing method. 24. The method of claim 19, wherein the method comprises an automated nucleic acid isolation, purification and/or collection method. 25. A method of applying one or more fluids to a solid support comprising the steps of: a) inserting at least one bioprocessing cassette into the bioprocessing device, said bioprocessing cassette comprising: i) at least one bioprocessing chamber containing a solid support therein; ii) a plurality of mesoscale and/or microscale channels in fluid communication with said biological processing chamber; iii) a film or foil layer encasing the at least one bioprocessing chamber, the plurality of mesoscale and/or microscale fluid channels and at least one pump; and iv) a cartridge alignment guide configured to retain the bioprocessing cartridge in a cartridge holder of a bioprocessing device; and b) implementing a pumping sequence on the cartridge, wherein the pumping sequence includes entering one or more fluid addition cycles in which fluid is pumped from the one or more containers through one of the fluid flow channels and pump into the cavity; wherein the fluid added in any of said fluid circuits is the same as or different from any other of said fluid circuits. 26. The method of claim 25, wherein the pumping sequence further comprises each fluid addition cycle followed by a purge cycle comprising pumping the fluid in the cavity into a designated waste container. 27. The method of claim 25, wherein the pumping sequence further comprises entering a flow-through cycle after any fluid addition cycle, wherein the flow-through cycle comprises opening a valve in a fluid flow channel connected to the bottom of the chamber, Fluid is pumped from the bottom of the cavity through one or more fluid flow channels and into the top of the cavity. 28. The method of claim 25, further comprising initiating and terminating the pumping sequence with a programmable controller. 29. The method of claim 25, wherein the pumping sequences performed on each of said cassettes are performed simultaneously. 30. An automated bioprocessing system comprising: a) Biological treatment plant comprising: i) one or more cartridge slots, each slot configured to accommodate a bioprocessing cartridge; and ii) an automated control system configured to control at least one parameter associated with bioprocessing in one or more bioprocessing cartridges, and b) one or more bioprocessing cartridges comprising i) at least one bioprocessing chamber configured to accommodate a solid support; ii) a plurality of mesoscale and/or microscale process fluid channels in fluid communication with the bioprocessing chamber via at least one pump, wherein the at least one pump is included in or on the bioprocessing cartridge; iii) a film or foil layer encasing the at least one bioprocessing chamber, the plurality of mesoscale and/or microscale fluid channels and the at least one pump; and iv) A cartridge alignment guide configured to retain the bioprocessing cartridge in a cartridge holder of a bioprocessing device.